Session 4: An Introduction to Polyketide Assembly Lines

00:00:07.09 Greetings.
00:00:08.25 This lecture,
00:00:10.13 or this trilogy of lectures,
00:00:13.12 is on the assembly-line biosynthesis
00:00:16.19 of a class of antibiotics
00:00:19.06 called the polyketide antibiotics.
00:00:22.28 Before I start,
00:00:25.08 I would like to share with you
00:00:27.11 the motivation
00:00:29.06 for recording a set of lectures
00:00:31.24 that I did in a similar setting
00:00:35.21 more than a decade ago.
00:00:39.23 There's three things that have changed.
00:00:42.26 The first one is that
00:00:45.11 the field of polyketide antibiotic biosynthesis
00:00:48.24 has moved forward quite a bit
00:00:51.14 over the past decade
00:00:53.10 and I thought it would be helpful for you
00:00:55.16 to know what has changed.
00:00:57.21 The second one is that the world of science,
00:01:00.15 and biochemistry in particular,
00:01:02.14 in which this topic sits,
00:01:04.29 has moved significantly too,
00:01:08.15 and so it would be helpful for you
00:01:10.29 to place what is new and important in this field
00:01:15.11 in the broader context of biochemistry.
00:01:18.19 And the third one, of course,
00:01:20.12 is I've gotten ten years older.
00:01:23.03 And while the latter generally
00:01:25.20 leads to more gray hair,
00:01:27.16 in my case considerably more
00:01:29.27 than what was there in the first version that I recorded,
00:01:34.08 it also gives somebody like myself,
00:01:37.04 who's been working on this problem
00:01:39.12 for more than two decades,
00:01:41.23 a chance to think about
00:01:44.01 where this field is going,
00:01:45.17 and I hope some of the things I share with you today
00:01:48.10 might give, especially the young people in the audience,
00:01:53.02 a chance to think about
00:01:55.09 where the field could be ten years from now
00:01:57.16 thanks to your own efforts.
00:01:59.19 Okay, so with that as a backdrop,
00:02:02.23 let me start with what you're looking at:
00:02:05.24 this picture of an automobile assembly line.
00:02:10.08 You all instantly recognize
00:02:13.10 what you're looking at in this picture.
00:02:16.19 You're looking at Henry Ford's contribution
00:02:20.07 to modern society.
00:02:22.21 And what you're seeing, more specifically,
00:02:25.09 is an assembly line
00:02:27.20 that builds cars
00:02:30.04 on a series of way stations
00:02:33.22 where at each way station
00:02:37.05 there are a set of catalysts,
00:02:39.01 human and mechanical,
00:02:42.03 that perform exquisite tasks
00:02:45.08 with a lot of sophistication,
00:02:48.03 but more or less
00:02:51.03 in a manner that is
00:02:54.03 oblivious about what is happening
00:02:56.23 upstream or downstream
00:02:59.24 of their way station.
00:03:02.14 And the genius of Henry Ford
00:03:05.04 lies in the modularity of this device.
00:03:09.28 So, the same assembly line
00:03:12.08 that builds a Ford Escort,
00:03:15.00 by changing a few things at the way stations
00:03:18.27 -- you could change some of the catalysts
00:03:21.03 that do certain operations
00:03:24.09 at a way station,
00:03:26.00 or you could change the inputs
00:03:28.26 at different points in the way station --
00:03:31.04 by those relatively simple and modular changes,
00:03:35.00 you can change the output,
00:03:37.04 which in one case could be a Fort Escort,
00:03:41.06 in another case might be a Lincoln Town Car
00:03:44.25 or your favorite automobile.
00:03:49.09 Now, nature has come up with a very similar strategy
00:03:53.05 to make a class of antibiotics
00:03:56.15 called the polyketide antibiotics,
00:03:59.28 and what I show you in this simple cartoon
00:04:03.10 is a prototypical example
00:04:06.25 of an assembly line
00:04:09.11 that's made up of a bunch of enzymes
00:04:11.13 and is responsible for making
00:04:14.10 a key intermediate
00:04:16.25 in the biosynthesis of the well-known antibiotic
00:04:19.29 Erythromycin.
00:04:22.01 This intermediate is called
00:04:23.24 6-Deoxyerythronolide B
00:04:27.08 and the assembly
00:04:28.24 that makes 6-Deoxyerythronolide B
00:04:31.14 is called the 6-Deoxyerythronolide B Synthase,
00:04:35.27 or DEBS for short.
00:04:38.25 And DEBS is made up
00:04:42.10 of three very large proteins.
00:04:46.20 You're seeing those three proteins
00:04:49.02 in cartoon forms on this slide.
00:04:53.15 Each protein,
00:04:55.11 you see the first protein made up of two modules
00:04:59.07 -- Modules 1 and 2 --
00:05:01.27 and an upstream bit
00:05:03.23 that we call the loading domain,
00:05:05.21 or LD for short.
00:05:08.00 And this protein is a homodimer.
00:05:11.02 You then have a second protein
00:05:13.29 that is also a homodimer
00:05:15.27 and is made up of two more modules of catalysts,
00:05:19.22 and then a final protein
00:05:21.26 that is made up of two additional modules
00:05:25.01 and another catalyst
00:05:27.11 called the TE, or thioesterase for short.
00:05:31.00 So this alpha2-beta2-gamma2 hexamer
00:05:36.17 that makes up this assembly line called DEBS,
00:05:41.23 has a molecular mass
00:05:45.07 of a little bit over 2 million daltons.
00:05:48.24 For those of you who don't know what 2 million daltons
00:05:51.24 buys you in biochemistry,
00:05:54.10 that's about the size of a ribosome.
00:05:57.08 And so, as a point of curiosity,
00:06:00.13 you may wish to acknowledge
00:06:03.01 that it takes nature a 2 million dalton machine
00:06:07.04 to make an antibiotic
00:06:09.04 whose job it is to gum up
00:06:11.03 the other 2 million dalton machine.
00:06:14.10 The rest of my lecture
00:06:15.29 is gonna be focused
00:06:18.10 on this assembly line DEBS.
00:06:21.05 Now, DEBS is an assembly line
00:06:24.25 that uses a bunch of precursors
00:06:28.16 that are available in metabolism.
00:06:31.15 You all are familiar with precursors
00:06:34.08 such as acetyl coenzyme A (acetyl-CoA)
00:06:38.15 or malonyl coenzyme A.
00:06:42.01 What you're seeing in this slide
00:06:44.14 are subtle variants of acetyl coenzyme A
00:06:49.00 and malonyl coenzyme A.
00:06:51.13 You're seeing a precursor
00:06:53.22 called propionyl coenzyme A
00:06:55.27 on the far left of this slide,
00:06:58.24 and the central precursor
00:07:00.27 that feeds into each of the six modules
00:07:05.15 of the assembly line
00:07:07.12 is a variant of malonyl coenzyme A
00:07:09.27 called methylmalonyl coenzyme A.
00:07:12.16 And so nature crafts this product,
00:07:16.23 6-Deoxyerythronolide B,
00:07:19.29 out of one equivalent of propionyl coenzyme A,
00:07:25.07 six equivalents of methylmalonyl coenzyme A,
00:07:29.28 and six equivalents of NADPH,
00:07:33.05 which you all know
00:07:34.29 is a reducing equivalent in biology.
00:07:39.16 And the way these precursors
00:07:42.08 come together to make
00:07:45.10 this molecule 6-Deoxyerythronolide B
00:07:49.09 that you're seeing to the far right of this assembly line
00:07:53.14 is by a mechanism
00:07:56.01 where you have propionyl coenzyme A
00:07:59.17 that starts the assembly line,
00:08:01.26 that primes the assembly line,
00:08:04.14 and through incremental addition of precursors,
00:08:08.11 you have at each module
00:08:11.22 on the assembly line,
00:08:13.08 you have further elaboration
00:08:15.21 of the precursor
00:08:18.04 to give you a highly complex product
00:08:22.14 at the end
00:08:24.15 called 6-Deoxyerythronolide B.
00:08:27.08 And this assembly line was discovered independently
00:08:30.29 by two research groups:
00:08:32.26 one at the University of Cambridge,
00:08:35.29 and another at Abbott Laboratories,
00:08:39.04 both who were working on this problem
00:08:42.01 about 25 years ago.
00:08:45.19 Now, just like Erythromycin,
00:08:48.18 there are a number of other complex antibiotics
00:08:52.24 that are made by this
00:08:58.02 assembly line strategy,
00:09:00.25 and you're seeing some of the who's who
00:09:03.12 among antibiotics
00:09:05.10 on this slide,
00:09:06.28 each of which is made
00:09:08.21 by a biosynthetic assembly line
00:09:10.27 that's similar, very similar,
00:09:12.20 to the kind that's used to make
00:09:15.02 6-Deoxyerythronolide B.
00:09:18.05 So, here's the outline
00:09:20.09 of what I have to say to you today.
00:09:24.02 I will first...
00:09:26.04 this module is going to focus
00:09:29.02 on three general overview topics
00:09:32.06 that I expect should be accessible
00:09:36.10 to anybody who has had,
00:09:38.12 or is concurrently taking,
00:09:40.28 a basic course in biochemistry.
00:09:43.25 I am going to tell you about
00:09:46.10 the evolutionary biology
00:09:49.00 of these assembly lines.
00:09:51.13 I will then talk a little bit about
00:09:53.15 the chemistry that happens
00:09:55.20 on the DEBS assembly line,
00:09:58.00 and then I'll give you an overview of
00:10:00.06 what this assembly line actually looks like
00:10:02.19 so you can put everything else in context.
00:10:06.21 In subsequently modules,
00:10:08.18 we'll talk about the tools the field uses
00:10:12.02 to study these assembly line enzymes
00:10:14.25 and then some properties
00:10:17.04 of these assembly lines
00:10:19.03 that represent cutting-edge topics
00:10:21.06 in modern research.
00:10:23.20 So let's start with the biology.
00:10:26.03 Back when we started working
00:10:28.01 on this assembly line,
00:10:30.10 which is way out here
00:10:32.13 perhaps even before then,
00:10:34.18 there was only one assembly line
00:10:36.27 that was known: DEBS.
00:10:39.26 And so you either worked on DEBS
00:10:41.23 or you did something else.
00:10:44.08 As you can see in this graph,
00:10:47.28 the world has changed significantly
00:10:50.10 over the past 20 years.
00:10:52.23 It had changed some,
00:10:54.09 but not a whole lot,
00:10:56.09 around the time I recorded
00:10:58.16 the earlier version of these lectures.
00:11:01.27 There were maybe a few tens
00:11:04.02 of these assembly lines
00:11:05.23 that had been painstakingly cloned
00:11:07.20 and sequenced
00:11:09.19 over the first 10 or 15 years on this slide,
00:11:14.00 and then something happened
00:11:16.02 around the mid-2000s.
00:11:18.12 As I'm sure most of you recognize,
00:11:20.18 that's around the time
00:11:22.07 it became relatively easy
00:11:24.01 to sequence genomes,
00:11:26.13 in particular bacterial genomes,
00:11:29.00 and since then the field
00:11:32.13 has exploded
00:11:34.10 in terms of the number of assembly lines
00:11:37.07 that are known to us
00:11:39.18 through sequence identity.
00:11:42.00 So, as of last summer,
00:11:44.22 there were close to 1000
00:11:47.12 distinct polyketide assembly lines
00:11:50.22 that had been cloned and sequenced,
00:11:52.28 and whose sequence had been deposited
00:11:55.10 in the database.
00:11:57.12 Now, what's important to note
00:11:59.09 about this slide
00:12:01.24 is a vast majority
00:12:04.07 of the assembly lines
00:12:05.27 whose sequence is available today
00:12:08.15 are what we call
00:12:10.09 orphan assembly lines.
00:12:12.09 Nobody really knows
00:12:14.04 what these assembly lines are doing in nature.
00:12:16.20 We just know their sequence.
00:12:18.18 And so we know they must be doing
00:12:20.28 something in nature,
00:12:22.12 or they probably are doing something,
00:12:24.26 but a vast majority,
00:12:26.23 about 80% of these assembly lines,
00:12:29.29 are begging for insights.
00:12:33.13 Here is an evolutionary tree
00:12:35.20 -- some of you may recognize it
00:12:37.13 as looking like a dendrogram --
00:12:39.21 of about 50 of the known
00:12:44.00 polyketide assembly lines
00:12:45.22 that have been sequenced to date.
00:12:48.07 And I don't expect you to read this slide
00:12:50.24 in detail,
00:12:53.10 but for those of you who have
00:12:55.26 heard about polyketide antibiotics,
00:12:58.06 to the right of this slide
00:12:59.26 what you're seeing are names
00:13:01.27 like Macrolides or Macrolide antibiotics,
00:13:06.05 FKBP-binding antibiotics
00:13:08.10 like FK-506 and Rapamycin,
00:13:11.10 Polyether antibiotics
00:13:13.09 that are frequently used in veterinary medicine.
00:13:16.09 These are...
00:13:18.00 Ansamycins, which include Rifamycin,
00:13:21.00 the front-line antibiotic to treat tuberculosis.
00:13:25.09 These are all antibiotics
00:13:28.02 that represent the who's who of infectious diseases,
00:13:32.09 cancer chemotherapy,
00:13:34.03 and related disease states.
00:13:37.01 These are a few examples
00:13:38.22 of polyketide assembly lines
00:13:40.21 whose sequence we know today.
00:13:44.07 But these are only a small fraction
00:13:47.10 of the polyketide assembly lines
00:13:51.28 whose existence we know of today.
00:13:54.14 So, what you're seeing on the far left
00:13:57.02 of this slide
00:13:59.23 is another family tree,
00:14:02.06 another dendrogram,
00:14:04.09 that I'm almost certain nobody can read
00:14:07.00 and I don't expect you to.
00:14:09.07 The point I want to leave you with
00:14:11.17 is that this vast pool
00:14:14.05 of orphan assembly lines
00:14:16.19 represents a really interesting starting point
00:14:20.04 for a field that's looking forward,
00:14:23.27 because the known polyketide assembly lines
00:14:28.01 today represent only small fractions
00:14:32.16 of this overall dendrogram.
00:14:35.11 There are large swaths of this family tree
00:14:39.05 where we know nothing about
00:14:41.05 what these polyketide synthases are doing.
00:14:44.27 And here's just one interesting factoid:
00:14:47.16 a lot of people who look at this field think,
00:14:50.25 "Oh, these are antibiotics biosynthetic enzymes,
00:14:53.11 they exist in bacteria."
00:14:55.20 That's true; many, perhaps most of these
00:14:59.05 antibiotic assembly lines exist in bacteria.
00:15:02.22 But this arrow down toward the bottom
00:15:06.11 of this slide
00:15:08.08 points to a small clade
00:15:10.11 in this very large
00:15:13.13 collection of orphans
00:15:16.00 that actually is encoded
00:15:18.12 by a bunch of worms.
00:15:20.19 And if you look a little bit further
00:15:22.22 at some of these assembly lines,
00:15:25.01 they seem to be making
00:15:27.06 some fairly complicated antibiotics.
00:15:29.14 Again, I want to emphasize
00:15:31.00 we don't know what these assembly lines
00:15:32.29 are making for certain,
00:15:34.23 but we can be reasonably confident
00:15:36.25 that these assembly lines
00:15:38.17 are making something very complex,
00:15:40.18 and they probably are doing so
00:15:42.16 for the benefit of the worms,
00:15:44.29 and we don't understand
00:15:48.01 what or how.
00:15:50.17 So, this field of polyketide biosynthesis,
00:15:54.22 one of the major things
00:15:56.19 that has happened is,
00:15:59.00 back when I recorded this in the past,
00:16:03.13 for any one of these assembly lines,
00:16:06.10 if you wanted the genetic information,
00:16:09.04 the blueprint for these assembly lines,
00:16:11.28 that would be close to an entire PhD thesis.
00:16:16.02 Today, you can get
00:16:19.01 thousands of these assembly lines
00:16:21.04 essentially for the price of free.
00:16:25.11 These exist in the database
00:16:27.05 and you can do whatever you want.
00:16:29.12 And so there's two major challenges
00:16:31.20 for the field going forward,
00:16:33.14 starting with this embarrassment of riches.
00:16:37.05 The first one is to develop the knowledge
00:16:40.07 that can help us decode
00:16:42.19 what these assembly lines
00:16:44.06 are doing in nature,
00:16:46.07 because that insight might let us
00:16:49.02 exploit the products of these orphan assembly lines
00:16:52.11 for interesting medical
00:16:54.07 and/or other applications.
00:16:57.04 The second thing
00:16:58.25 that one could hope the field can deliver
00:17:01.02 over the coming decade
00:17:03.03 is the insights
00:17:05.19 that might allow us to start
00:17:08.00 with these 1000 or 2000 assembly lines
00:17:11.05 and scramble them in ways
00:17:13.24 to make molecules that nature
00:17:17.02 probably didn't bother trying out,
00:17:19.05 or maybe nature tried out
00:17:20.18 but didn't find much use for,
00:17:22.21 but humanity could find use for.
00:17:25.29 And that represents another
00:17:27.17 important goal in the field.
00:17:30.28 Okay, so with that as a biological background,
00:17:33.15 let's switch to the chemistry
00:17:35.10 that happens on one of these
00:17:37.10 polyketide assembly lines,
00:17:39.07 namely DEBS.
00:17:40.22 So, I introduced you to this assembly line,
00:17:43.17 DEBS,
00:17:45.04 that makes this intricate molecule
00:17:47.06 to the right
00:17:48.21 called 6-Deoxyerythronolide B.
00:17:50.24 Perhaps the best way for me
00:17:53.10 to give you a sense of the actual
00:17:55.02 enzymatic chemistry
00:17:56.24 that's happening on this assembly line
00:17:59.03 is by zeroing in
00:18:01.13 on one of those modules,
00:18:03.18 and I've boxed Module 3
00:18:06.09 for illustration purposes,
00:18:08.24 and let's take a close look
00:18:10.29 at what's happening in Module 3,
00:18:13.21 as well as its two interfaces
00:18:16.07 with its neighboring modules,
00:18:18.07 Module 2 and Module 4, respectively.
00:18:21.23 Because if we can understand
00:18:23.22 what Module 3 does
00:18:25.28 in the overall biosynthetic process,
00:18:29.03 how it talks to Module 2
00:18:31.25 and how it hands off its produc
00:18:34.28 t to Module 4,
00:18:36.20 then the rest of this pathway
00:18:39.05 becomes relatively straightforward
00:18:41.06 to understand.
00:18:43.08 So, in order to explain to you
00:18:45.16 what Module 3 does,
00:18:48.09 we need to go into
00:18:50.08 a higher level of granularity,
00:18:52.16 and this is where I have introduced
00:18:54.16 a few acronyms in the slide
00:18:57.10 for those of you who are still paying attention,
00:19:00.06 you've started to see,
00:19:01.25 in what I had earlier on called
00:19:04.14 Modules 2, 3, and 4,
00:19:06.11 the appearance of some acronyms
00:19:09.07 'KS', 'AT', 'KR', 'ACP', and so on.
00:19:16.00 For the initiated,
00:19:18.14 these are the quintessential
00:19:22.05 catalytic domains
00:19:24.04 that one finds in all of these
00:19:26.03 polyketide assembly lines.
00:19:28.12 For the uninitiated in you,
00:19:30.18 in the lower-left corner of this slide,
00:19:33.13 and in all subsequent slides
00:19:35.08 where I use these kinds of acronyms,
00:19:38.11 I'll keep a key
00:19:40.02 so that you can reference quickly
00:19:42.18 what kind of enzyme or protein
00:19:45.09 I'm talking about,
00:19:46.23 and so when you see
00:19:48.15 the letters KS in one of these modules,
00:19:52.15 you know I am talking about
00:19:54.25 a ketosynthase,
00:19:56.25 and I'll introduce you to the enzymology
00:19:59.09 of a ketosynthase
00:20:01.06 in a moment.
00:20:02.20 Similarly, when you see the letters
00:20:04.21 AT, I'm talking about an acyltransferase.
00:20:08.09 When you see the letters
00:20:10.00 KR, I'm talking about a ketoreductase,
00:20:14.03 and so on and so forth.
00:20:17.03 So in order to understand
00:20:18.29 what Module 3 does,
00:20:21.09 what we're gonna do
00:20:22.20 is we're gonna peel away
00:20:24.24 everything else in the assembly line
00:20:26.15 except for Module 3,
00:20:29.28 the ACP, or the acyl carrier protein
00:20:33.24 from the upstream module,
00:20:35.20 because that's the domain of the upstream Module 2
00:20:40.05 that donates the polyketide chain
00:20:42.12 into Module 3,
00:20:44.15 and the KS, or ketosynthase
00:20:47.19 from Module 4, because that is the recipient
00:20:50.20 of the product of Module 3.
00:20:53.27 And now we can look
00:20:56.03 at the catalytic cycle
00:20:58.16 associated with Module 3.
00:21:00.26 So we're gonna start
00:21:02.22 from the state of this module
00:21:05.12 that is shown at 10 o'clock
00:21:08.18 on this catalytic cycle,
00:21:11.02 where you see that the Module 3 itself
00:21:14.22 is empty;
00:21:16.06 it has no precursors bound to it.
00:21:19.02 There is a substrate
00:21:22.03 that is bound to the upstream
00:21:24.22 acyl carrier protein,
00:21:26.17 which is ready to come into Module 3,
00:21:29.04 and Module 3,
00:21:31.05 from its previous catalytic cycle,
00:21:32.29 has donated its product
00:21:35.12 to the next module,
00:21:37.13 Module 4.
00:21:39.12 So, starting from that point,
00:21:41.15 the first chemical step that needs to occur
00:21:44.10 is a translocation event.
00:21:47.03 In this event,
00:21:48.26 the growing polyketide chain,
00:21:51.12 which was a triketide
00:21:54.06 on the acyl carrier protein
00:21:57.02 of the upstream module,
00:21:59.16 has been moved into
00:22:02.22 the Module 3
00:22:04.16 and is now bound
00:22:06.17 to the ketosynthase
00:22:08.12 through what you might recognize
00:22:10.06 as a thioester linkage.
00:22:13.04 So the substrate
00:22:15.05 was an acyl protein carrier-bound thioester,
00:22:18.18 and the product is also a thioester,
00:22:21.17 but now it's bound in the underbelly
00:22:24.27 of Module 3
00:22:26.25 at the ketosynthase active site.
00:22:29.27 That reaction, from here on out,
00:22:32.15 we're gonna talk about
00:22:34.06 as a translocation event,
00:22:36.04 because the chain has been translocated
00:22:38.20 from one module to the next.
00:22:42.13 The next event in the catalytic cycle
00:22:44.17 is an acyl transfer event.
00:22:47.24 This is when Module 3
00:22:50.04 makes a critical choice
00:22:52.03 about what precursor
00:22:54.02 it is gonna pick from the cell soup,
00:22:56.20 from the metabolic pool of precursors
00:22:59.14 that exists in the cell,
00:23:01.20 and bring it inside
00:23:03.19 so it can catalyze
00:23:05.22 the next set of operations,
00:23:08.02 and this event we're gonna call
00:23:09.29 acyl transfer.
00:23:12.03 It is catalyzed by the acyltransferase,
00:23:15.15 or AT for short,
00:23:17.21 and you'll see Module 3
00:23:19.12 has one of those domains in it.
00:23:22.08 And the acyltransferase of Module 3
00:23:25.17 has picked
00:23:27.16 a methylmalonyl extender unit
00:23:30.15 from a coenzyme A-bound precursor
00:23:34.12 and transferred that methylmalonyl extender unit
00:23:38.00 onto the ACP, or acyl carrier protein,
00:23:42.25 of that module.
00:23:45.15 So now,
00:23:47.15 we are at about 3pm
00:23:49.28 of this catalytic cycle.
00:23:52.06 You have an electrophilic,
00:23:54.10 growing polyketide chain
00:23:56.13 attached as a thioester
00:23:58.28 to the ketosynthase.
00:24:01.05 You have a nucleophile,
00:24:02.27 the methylmalonyl extender unit
00:24:05.16 that is attached to the acyl carrier protein,
00:24:08.29 and the stage is set up
00:24:11.03 for the next reaction,
00:24:13.06 which we're gonna call
00:24:15.05 the elongation reaction.
00:24:17.08 This is where
00:24:19.03 you see a carbon-carbon bond
00:24:21.25 having formed
00:24:23.24 between this 3-carbon unit
00:24:25.21 and the rest of the chain
00:24:27.21 that you see
00:24:29.14 that came from the upstream module.
00:24:32.11 So, this reaction
00:24:34.06 is the elongation step
00:24:37.09 and it is, for those of you who are considering
00:24:40.08 the overall thermodynamics of the process,
00:24:43.11 this is the key energy-giving step
00:24:47.00 in the process.
00:24:49.03 From a thermodynamic perspective,
00:24:50.24 it's this release of CO2 in the step
00:24:53.27 that drives this overall enzymatic process forward.
00:25:00.09 The next reaction,
00:25:02.09 that I call chain modification,
00:25:05.03 is a variable set of operations
00:25:07.13 that this module does.
00:25:09.29 In this particular case,
00:25:12.07 all that the enzyme has done
00:25:15.06 is it's catalyzed
00:25:17.07 a racemization of the C2 carbon
00:25:21.13 of the newly elongated polyketide chain.
00:25:25.22 So the chain elongation step
00:25:30.21 involved what is known as an
00:25:33.16 inversion of stereochemistry.
00:25:36.02 The (2S)-methylmalonyl extender unit
00:25:39.21 stereochemistry got inverted,
00:25:42.24 and you have the product that is formed.
00:25:45.26 And in the modification step
00:25:51.07 that second carbon in the growing polyketide chain
00:25:54.22 is scrambled
00:25:57.02 to give you a racemic center.
00:25:59.26 That step is catalyzed
00:26:02.08 by the enzyme that I have designated in this module
00:26:05.14 as a KR0.
00:26:08.11 Now, for those of you who are
00:26:10.04 paying attention to the key I have
00:26:13.01 in the lower-left corner,
00:26:15.04 you're wondering
00:26:16.24 why did I call a racemase
00:26:19.06 a ketoreductase.
00:26:21.09 That will become apparent to you
00:26:23.08 as we go further in this lecture.
00:26:27.15 Once the modification occurs,
00:26:29.25 now you have both different flavors of stereochemistry available
00:26:36.14 and the chain is ready to move
00:26:38.17 into the next module,
00:26:40.20 which selective chooses
00:26:43.01 only one of those two diastereomers
00:26:46.16 to process further,
00:26:48.10 well the other one can be racemized again
00:26:51.12 by that ketoreductase-like racemase
00:26:54.14 to give you additional precursors
00:26:57.01 that can be moved forward.
00:26:59.10 And so the take-home message from this slide
00:27:02.28 is that a catalytic cycle
00:27:05.20 of a polyketide assembly line module
00:27:08.25 comprises of
00:27:11.29 two translocation events,
00:27:13.26 one from the upstream module,
00:27:15.16 one to the downstream module,
00:27:18.06 an acyl transfer that picks a building block,
00:27:21.19 a chain elongation event,
00:27:24.09 and one or more modification events
00:27:27.09 that leads to diversity generation
00:27:30.07 on the growing polyketide chain.
00:27:33.05 And now that you understand
00:27:35.01 what Module 3 does,
00:27:37.17 it becomes relatively easy for you
00:27:39.18 to see how each of the six modules
00:27:43.00 of the 6-Deoxyerythronolide B Synthase
00:27:47.00 perform a set of catalytic operations,
00:27:51.02 in each case
00:27:52.22 on a methylmalonyl extender unit,
00:27:55.06 and a unique incoming polyketide chain,
00:27:58.15 to give you the product
00:28:00.19 that comes out of this assembly line.
00:28:04.02 Okay, so now that you understand the chemistry
00:28:07.08 of this assembly line,
00:28:09.07 let's talk a little bit about the structure.
00:28:11.28 Now, clearly, I'm showing you...
00:28:14.14 you must recognize that even though I show you
00:28:17.02 this assembly line
00:28:18.17 in cartoon form as I showed you in this slide,
00:28:21.13 this is not what the system looks like in nature,
00:28:24.00 and many of you are probably already asking this question:
00:28:26.29 what does this assembly line
00:28:29.15 look like in three dimensions?
00:28:32.05 So, this is what we know today
00:28:35.12 about the 6-Deoxyerythronolide B [synthase].
00:28:40.00 So on the top of this slide
00:28:41.19 I show you that same assembly line
00:28:43.23 that I showed you earlier on,
00:28:46.01 with all those active sites
00:28:48.06 labeled the same way,
00:28:50.15 but now what I have highlighted
00:28:53.21 in green are the portions of the assembly line
00:28:58.13 whose atomic structures have been solved,
00:29:01.22 primarily by X-ray crystallography,
00:29:05.04 but also using NMR.
00:29:09.07 What you can see over here is,
00:29:11.22 we know today the atomic structures
00:29:14.10 of about a quarter
00:29:16.11 to a third of this overall assembly line.
00:29:19.17 These structures of the different pieces
00:29:22.11 that I've highlighted in green-blue
00:29:25.11 have been solved
00:29:28.16 by first extracting these pieces
00:29:30.18 out of the assembly line
00:29:33.08 and then solving the structures
00:29:36.22 of these pieces.
00:29:38.29 What is important to recognize is that
00:29:42.19 the DEBS assembly line
00:29:45.12 has a very strong
00:29:47.21 repetitive characteristic.
00:29:50.08 So, different active sites
00:29:52.14 occur again and again
00:29:54.12 in the assembly line.
00:29:56.07 What you will see is
00:29:58.03 there is a ketosynthase, KS,
00:30:00.01 that is associated with each of the six core modules
00:30:04.01 of the assembly line,
00:30:05.29 as is an acyltransferase, or AT,
00:30:08.20 or an ACP.
00:30:10.18 So, what you have are
00:30:13.01 domains that have homologues,
00:30:15.19 and these are very homologous domains,
00:30:17.28 so any two of the ketosynthases
00:30:20.16 in the 6-Deoxyerythronolide B Synthase
00:30:23.20 have upwards of 50% identity.
00:30:26.17 And through this divide and conquer approach,
00:30:30.17 one can therefore derive
00:30:33.05 a fairly good insight
00:30:35.10 into what the atomic structures
00:30:37.14 of any of the individual domains
00:30:40.15 within the assembly line are.
00:30:42.16 So, we have atomic structures
00:30:44.23 of at least one prototypical domain
00:30:48.01 of all of these active sites
00:30:51.22 that comprise the DEBS enzymatic assembly line.
00:30:55.25 So, that information...
00:30:57.19 starting from that information,
00:31:00.02 one can now start to build models
00:31:02.24 for what an actual catalytic module
00:31:05.29 might look like.
00:31:08.26 This cartoon that I show you here
00:31:12.12 is our best guess...
00:31:15.08 so at the bottom of this cartoon
00:31:17.05 you're seeing, in color,
00:31:19.19 the DNA arrangement of the domains
00:31:23.29 that make up one of these modules
00:31:26.25 that contains a ketosynthase,
00:31:28.24 an acyltransferase,
00:31:31.01 a ketoreductase,
00:31:32.14 acyl carrier protein,
00:31:34.02 and so on.
00:31:36.00 And at the same time
00:31:37.22 what you're seeing on the top
00:31:39.19 is a model
00:31:41.21 for what this module might look like
00:31:44.12 in three dimensions.
00:31:46.15 Some aspects that you're seeing in this model,
00:31:49.08 in the structural model,
00:31:52.08 are hard facts,
00:31:53.27 because we have actual X-ray crystallographic structures
00:31:56.29 or NMR structures
00:31:58.26 of the pieces,
00:32:00.17 but the relative orientations of these...
00:32:03.05 so for example,
00:32:04.27 the relative orientation of the pale blue part on the top
00:32:08.10 and the lower butterfly-like structure
00:32:12.13 is somewhat speculative,
00:32:15.05 and it's been derived
00:32:17.03 primarily through models
00:32:19.19 that compare this polyketide synthase
00:32:22.26 with the vertebrate fatty acid synthase
00:32:25.15 that is a homologue of this module,
00:32:27.24 and whose structure has been solved.
00:32:31.16 Now, how might one get additional data
00:32:34.20 on what these modules might look like?
00:32:37.12 This is where one uses
00:32:39.16 lower resolution methods,
00:32:41.20 in our case over here
00:32:43.22 we've used SAXS,
00:32:46.05 or small-angle X-ray scattering.
00:32:48.27 The graph that I show you to the left
00:32:52.08 is a typical plot
00:32:54.23 that one gets of scattering intensity
00:32:57.07 against scattering angle,
00:32:59.13 and from that data
00:33:01.08 one can get information
00:33:03.09 about the size and shape
00:33:06.05 of the protein
00:33:08.05 that has been subjected to SAXS analysis.
00:33:11.10 And from that information
00:33:13.03 about size and shape,
00:33:14.28 one can derive
00:33:16.28 lower-resolution, but still useful models
00:33:19.29 for what the module might look.
00:33:22.11 And what you're seeing in this slide
00:33:24.05 suggests that the model I showed you
00:33:26.17 in the earlier slide,
00:33:28.09 which was derived from homology
00:33:29.28 with the fatty acid synthase,
00:33:31.19 isn't too far off-base.
00:33:34.13 You can use SAXS also
00:33:37.27 to look at larger pieces of the assembly line.
00:33:41.06 So, here we're looking at the SAXS data,
00:33:47.15 scattering data
00:33:49.02 derived from a very large
00:33:51.10 two-module protein
00:33:54.09 that has a homodimeric molecular mass
00:33:56.23 on the order of 650 kiloDaltons.
00:34:00.05 Again, the key take-home message
00:34:02.21 that you wanna take from this slide is
00:34:05.10 that there is a very defined three-dimensional architecture
00:34:09.05 that one can predict
00:34:11.13 for these assembly lines,
00:34:13.21 or in this case
00:34:15.05 two adjacent modules of the assembly line.
00:34:17.29 And if you put this kind of model building together,
00:34:21.14 what you can get is insight into,
00:34:24.29 or at least a working model
00:34:29.09 for what the overall assembly line
00:34:31.12 might look like.
00:34:33.09 So what you're seeing in this cartoon
00:34:35.06 is our best guess, today,
00:34:37.12 of what the assembly line looks like.
00:34:40.07 There's a Module 1,
00:34:42.08 denoted as M1,
00:34:44.03 followed by a Module 2,
00:34:46.12 and you're seeing a zigzag type of a structure
00:34:51.05 that gives you a sense of what this 2 million Dalton
00:34:53.18 assembly line looks like.
00:34:56.00 So I will stop over here.
00:34:58.07 Hopefully this gives you a basic overview
00:35:00.09 of what these assembly line polyketide synthases are,
00:35:05.23 what chemistry they do,
00:35:07.16 and what they look like,
00:35:09.11 or at least our best guess, today,
00:35:11.04 of what they look like.
00:35:13.02 In subsequent modules,
00:35:14.13 we'll talk about other aspects of
00:35:17.10 these remarkable machines in nature.
00:35:19.16 Thank you.

00:00:08.07 Greetings.
00:00:09.18 My name is Chaitan Khosla
00:00:11.09 and I'm a professor at Stanford University,
00:00:15.19 and this is part two
00:00:18.01 of the trilogy of my lectures
00:00:19.28 on assembly line polyketide biosynthesis.
00:00:24.11 In the previous lecture
00:00:25.27 I introduced you to the evolutionary biology
00:00:30.15 of these remarkable assembly lines,
00:00:33.00 the chemistry that happens
00:00:34.10 on these assembly lines,
00:00:36.07 and I gave you a general idea
00:00:38.05 of what these assembly lines look like.
00:00:42.02 So, we looked at the
00:00:44.12 6-Deoxyerythronolide B,
00:00:47.03 or DEBS,
00:00:48.22 synthase,
00:00:50.11 that is responsible for making
00:00:52.23 this precursor of erythromycin.
00:00:57.21 I ended the previous lecture
00:01:00.15 by giving you a sense of what
00:01:02.18 we think this assembly line looks like
00:01:04.22 and how that insight was derived.
00:01:08.21 What I'm gonna start this module with
00:01:12.02 is an introduction to the kinds of tools
00:01:14.24 we use to interrogate
00:01:17.01 the biochemistry
00:01:19.06 of these remarkable assembly lines.
00:01:22.12 So,
00:01:25.13 these assembly lines exist,
00:01:27.27 as we discussed in the first lecture,
00:01:30.22 in relatively esoteric sources.
00:01:33.17 They usually come from bacteria
00:01:35.22 whose names many of us have a hard time spelling,
00:01:39.11 or sometimes even worms,
00:01:41.25 or often times just sequence information
00:01:45.04 that was derived from DNA
00:01:47.04 that was collected from some place.
00:01:50.16 In order to study these systems,
00:01:53.07 one of the first sets of tools
00:01:55.14 that we developed
00:01:57.24 was to be able to take these
00:01:59.15 very complex metabolic pathways,
00:02:02.12 like the DEBS metabolic pathway,
00:02:04.26 and put them into
00:02:08.00 genetics-friendly microorganisms;
00:02:10.17 hosts like E. coli.
00:02:13.21 So, today you can make
00:02:16.20 6-Deoxyerythronolide B
00:02:19.15 in E. coli
00:02:21.07 by growing it in the presence of
00:02:24.12 glucose as a source of energy
00:02:26.11 and propionic acid
00:02:28.00 as a source of all the carbon
00:02:30.05 that's used to make the product,
00:02:32.04 so long as the recombinant E. coli
00:02:34.11 contains DNA
00:02:36.26 that instructs for the biosynthesis
00:02:39.10 of those three very large proteins
00:02:42.09 that comprise DEBS.
00:02:44.18 This is,
00:02:46.16 for obvious reasons,
00:02:48.18 a very powerful tool
00:02:50.15 to interrogate DEBS
00:02:52.06 because, now, if I have a bacterium
00:02:54.29 that makes my product for me,
00:02:57.09 I can go in there
00:02:59.15 for the price of a $200 kit,
00:03:02.12 manipulate the DNA
00:03:04.12 that encodes this assembly line
00:03:06.19 and ask,
00:03:08.06 what are the consequences of this assembly line?
00:03:11.03 And these tools have been
00:03:13.02 in our armamentarium
00:03:15.09 for the better part of the past two decades.
00:03:17.20 My previous generation of lectures
00:03:20.01 talked quite a bit about these tools,
00:03:22.11 so I won't spend a lot of time
00:03:24.01 doing so again,
00:03:26.05 but these tools
00:03:28.12 have played a critical role
00:03:30.03 in our understanding
00:03:31.21 of the biochemistry
00:03:33.01 of these assembly lines.
00:03:34.17 Now, what is more challenging,
00:03:37.12 but is perhaps arguably more important is,
00:03:41.28 if you want to study this remarkable enzymatic assembly line,
00:03:46.01 you'd like to be able to peel off the wrapper
00:03:49.28 that surrounds these remarkable proteins.
00:03:53.08 That is easier said than done,
00:03:55.20 but today, we can reconstitute
00:03:58.20 the entire 6-Deoxyerythronolide B synthase
00:04:02.15 from purified proteins.
00:04:05.06 What I show you on the lower-left corner
00:04:09.09 is a protein gel, an SDS-PAGE,
00:04:12.27 that shows five proteins.
00:04:17.20 The two proteins to the far right
00:04:21.01 are the second
00:04:23.23 and the third protein
00:04:26.15 of the erythromycin assembly line.
00:04:30.20 And each of them, as you can tell,
00:04:33.12 has a monomeric molecular mass
00:04:36.01 that exceeds 300 kilodaltons.
00:04:40.01 The third protein,
00:04:41.22 which is the first of these proteins,
00:04:45.03 could not be expressed
00:04:46.23 for love or money
00:04:48.14 in E. coli
00:04:50.13 in a form that gave adequate yields
00:04:53.07 of pure protein
00:04:54.28 to study biochemically,
00:04:56.28 and so we had to break it up
00:04:58.26 into three pieces,
00:05:01.04 which are shown in the
00:05:02.24 first three [lanes] of this gel,
00:05:05.14 and purify those pieces independently.
00:05:10.08 And now you can put those three pieces
00:05:13.14 together with the other two proteins
00:05:16.03 to make a cocktail of proteins
00:05:19.25 that, in the presence of appropriate substrates
00:05:23.06 -- propionyl coenzyme A,
00:05:25.11 NADPH,
00:05:27.04 and we don't use methylmalonyl coenzyme A itself,
00:05:31.01 instead we use an in situ enzymatic generation method
00:05:35.22 for methylmalonyl coenzyme A
00:05:38.00 where we use free methylmalonic acid,
00:05:41.01 coenzyme A,
00:05:42.24 and an enzyme called malonyl-CoA synthetase --
00:05:46.07 and so when you put these five proteins,
00:05:49.14 which have been purified,
00:05:51.13 together with all these precursors
00:05:53.26 in a test tube,
00:05:55.21 you see 6-Deoxyerythronolide B.
00:05:58.24 And what I show you on the lower right
00:06:01.07 is a mass spectrum of the product
00:06:04.00 that has been synthesized
00:06:05.29 in a biochemical equivalent
00:06:08.02 of an earth/air/water/fire type
00:06:10.19 of an experiment.
00:06:12.16 What this allows us now to do
00:06:14.24 is to probe this machine
00:06:17.05 with all the power
00:06:19.19 that you're used to using
00:06:21.24 to study your favorite enzyme
00:06:24.27 once you've purified it to homogeneity.
00:06:28.06 So what I show you in this
00:06:30.13 is a very simple graph
00:06:33.05 that gives you a sense
00:06:35.03 that we can turnover
00:06:36.26 this entire assembly line
00:06:38.27 in a test tube,
00:06:40.18 with a rate constant
00:06:42.15 that's approximately about 1/min.
00:06:47.10 So, approximately once every minute
00:06:49.17 this assembly line is releasing
00:06:51.21 6-Deoxyerythronolide B
00:06:53.22 in a test tube that is presented with the appropriate precursors,
00:06:57.27 and that is roughly the rate
00:06:59.24 we might expect this assembly line
00:07:01.18 to be working at
00:07:03.14 inside a cell.
00:07:05.06 I also wanna point out,
00:07:06.24 as the inset shows,
00:07:08.21 this assay is remarkably efficient.
00:07:12.29 Every equivalent of 6-Deoxyerythronolide B
00:07:16.28 has stoichiometric mapping
00:07:19.17 to an equivalent
00:07:21.26 of the propionyl-CoA primer
00:07:24.01 that is used,
00:07:25.28 and uses six equivalents of NADPH,
00:07:29.08 whose consumption is being measured
00:07:31.05 in this simple spectrophotometric assay.
00:07:35.07 Okay, so we have to tools to be able to study
00:07:39.04 the entire assembly line
00:07:40.25 inside a recombinant E. coli-like cell.
00:07:44.06 We have the ability
00:07:46.00 to study the entire assembly line
00:07:48.01 in a purified, reconstituted form.
00:07:51.03 We also have the ability, today,
00:07:53.21 to study the individual steps
00:07:56.12 in the catalytic cycle of these modules
00:08:01.09 in isolation.
00:08:03.10 So, recall in the previous module,
00:08:05.29 I introduced you to some of the
00:08:08.12 core reactions
00:08:10.18 that occur at every module.
00:08:13.00 There's a reaction
00:08:14.19 that we call chain translocation,
00:08:16.24 where the chain moves
00:08:18.08 from the acyl carrier protein
00:08:20.04 in the upstream module
00:08:21.26 to the module that's receiving the chain,
00:08:25.09 and if we want to interrogate
00:08:27.07 just that reaction
00:08:29.15 for one module,
00:08:31.09 what we do is pull out that module
00:08:34.07 from the rest of the assembly line,
00:08:36.17 purify that to homogeneity,
00:08:39.23 present it with
00:08:43.01 a chemoenzymatically-derived acyl carrier protein
00:08:47.00 that has the growing polyketide chain substrate
00:08:51.01 bound to it,
00:08:53.03 and we put it into a test tube
00:08:55.19 so that the chain translocation event
00:08:58.13 -- the movement of that growing polyketide chain
00:09:01.23 into the module --
00:09:03.26 is the slow kinetic step,
00:09:06.00 and everything after that
00:09:08.03 that leads to the turnover of this module
00:09:10.21 is fast.
00:09:12.08 And so you can use
00:09:14.07 this kind of an assay
00:09:15.28 to interrogate,
00:09:18.01 using established kinetic paradigms,
00:09:20.23 that chain translocation step
00:09:23.10 of your favorite module
00:09:25.12 that you're interested in.
00:09:27.22 The same approach can also be used
00:09:31.03 to kinetically isolate the chain elongation event
00:09:34.11 that I introduced you to
00:09:36.03 in the earlier lecture.
00:09:38.18 So when we want to study chain elongation,
00:09:42.03 what we do is we take the module
00:09:44.22 whose elongation biochemistry
00:09:46.19 we want to study,
00:09:48.19 and we prepare just the ketosynthase
00:09:51.03 together with the acyltransferase
00:09:54.08 from that module
00:09:56.02 as one protein.
00:09:57.21 We produce its carrier protein,
00:10:00.04 its acyl carrier protein,
00:10:01.29 as another protein.
00:10:03.27 We put these two proteins together,
00:10:06.21 we present the two substrates
00:10:09.24 into this assay,
00:10:12.05 and we look for chain elongation,
00:10:15.00 which gives rise to the product.
00:10:17.19 And we do this under conditions
00:10:20.06 where the step we wanna probe,
00:10:22.07 the elongation step,
00:10:23.27 is the slow step, and everything else is fast.
00:10:27.20 You can do exactly the same thing
00:10:29.21 to probe the acyl transfer,
00:10:31.29 the selection of that building block
00:10:34.16 -- methylmalonyl coenzyme A-derived building block
00:10:38.12 from metabolism --
00:10:40.08 the same approach can also work over there.
00:10:42.20 And all of these assays are well-developed,
00:10:44.29 they're in the literature,
00:10:46.19 and you can use them to study
00:10:48.08 your favorite assembly line.
00:10:51.07 In addition to those core reactions
00:10:53.28 -- chain translocation,
00:10:55.21 chain elongation,
00:10:57.08 and acyl transfer --
00:10:58.23 I mentioned there are auxiliary reactions,
00:11:01.13 which I lumped under chain modification.
00:11:04.27 Those reactions include
00:11:06.22 ketoreductase-types of chemistries.
00:11:10.11 In this particular assay,
00:11:12.11 I'm adding the ketoreductase,
00:11:15.03 or KR,
00:11:16.26 as a stand-alone protein,
00:11:18.16 to the rest of my system
00:11:21.04 so that I can control the rate
00:11:22.27 at which that step occurs,
00:11:24.27 and I can look at the consequences
00:11:27.13 of putting one ketoreductase in my assay
00:11:30.04 as opposed to some other ketoreductase.
00:11:33.04 And that allows me to interrogate
00:11:35.28 the ketoreductase reaction.
00:11:38.00 You can do the same thing
00:11:40.04 at the level of the dehydratase reaction,
00:11:43.15 which follows after the ketoreductase reaction
00:11:47.29 in certain chain modification sequences.
00:11:52.00 So, all of these assays are also set up.
00:11:54.22 The point you need to recognize is that
00:11:57.13 you can probe through, again,
00:11:59.04 a divide-and-conquer approach,
00:12:01.00 the chemistry happening at any one of these steps
00:12:05.05 in the overall assembly line.
00:12:08.22 Using this combination of in vivo and in vitro tools,
00:12:14.02 there are a number of important problems
00:12:16.15 you can study.
00:12:17.28 In the remainder of this second module lecture,
00:12:21.16 I will talk to you about some examples
00:12:24.13 of questions, long-standing questions
00:12:26.26 in the field,
00:12:28.04 having to do with the specificity
00:12:29.28 of these assembly lines.
00:12:31.20 I'll give you two examples of those problems
00:12:33.22 because they have engineering implications.
00:12:36.21 And then in the next lecture we'll talk about
00:12:40.08 the assembly line mechanisms.
00:12:42.27 So, stereospecificity
00:12:46.01 is probably one of the most fascinating features
00:12:50.15 of these complex polyketide antibiotics
00:12:53.11 that these assembly lines make.
00:12:55.24 So, to the right,
00:12:57.14 you're seeing the 6-Deoxyerythronolide B product
00:13:01.16 of DEBS,
00:13:03.07 and for those of you who are looking at that now,
00:13:05.22 you're noticing that it has 10 stereocenters.
00:13:12.02 That is 2^10 possible chiral forms
00:13:17.08 of the same chemical formula,
00:13:20.01 or slightly more than 1000
00:13:22.12 of these chiral forms.
00:13:24.20 If you go to a fermentation plant
00:13:27.10 that makes erythromycin,
00:13:30.08 the large vat that produces erythromycin
00:13:33.23 has one out of those 1000+
00:13:38.04 stereochemical forms in it;
00:13:40.17 the one that I'm showing you.
00:13:43.00 I think most of you would recognize
00:13:45.01 that that is a really impressive feat
00:13:48.04 on the part of nature...
00:13:49.27 how it can program this assembly line
00:13:52.06 to give one, and only one
00:13:54.13 stereochemical outcome.
00:13:56.22 That is a problem
00:13:58.29 that we have quite a good understanding of
00:14:02.16 how that happens today.
00:14:05.08 I've cited some references on this slide,
00:14:09.04 and so I will summarize for you
00:14:11.07 what these references teach us
00:14:13.18 about how stereochemistry is controlled
00:14:17.07 by the DEBS assembly line.
00:14:20.00 So, of those 10 stereocenters,
00:14:25.00 one of them,
00:14:27.12 which is this stereocenter,
00:14:31.06 is generated by
00:14:34.28 this ketoreductase
00:14:37.28 in Module 3 of the assembly line,
00:14:40.19 that I introduced to you as that epimerase,
00:14:44.29 that looks like a ketoreductase
00:14:46.29 in my previous lecture.
00:14:49.06 This is the enzyme that is a homologue
00:14:52.02 of other ketoreductases,
00:14:53.18 but does no NADPH-dependent chemistry.
00:14:57.27 Instead, it epimerizes the C2 carbon atom
00:15:01.20 of the growing polyketide chain
00:15:04.28 that is lodged in Module 3
00:15:06.25 of the assembly line.
00:15:09.05 Of the remaining 9 stereocenters,
00:15:12.07 8 of those stereocenters
00:15:14.22 are shown in red,
00:15:17.09 and they are controlled
00:15:19.21 by the 3 red ketoreductases
00:15:23.02 and 1 blue ketoreductase
00:15:25.17 in Modules 1, 2, 5, and 6, respectively.
00:15:32.23 So, each of these 4 ketoreductases
00:15:37.25 controls 2 stereocenters apiece.
00:15:42.20 For the chemically initiated,
00:15:44.29 these enzymes are not just stereoselective,
00:15:48.22 they're also diastereoselective;
00:15:51.17 so they're setting 2 stereocenters at a time.
00:15:56.01 And these enzymes, we know...
00:15:58.23 these 4 enzymes we know, today,
00:16:01.13 are both necessary and sufficient
00:16:04.10 for the unique labeling...
00:16:08.05 for the unique identification
00:16:11.01 of those stereocenters.
00:16:13.21 The last stereocenter,
00:16:16.01 which is this stereocenter,
00:16:19.13 is at the 6 position,
00:16:22.01 is a more complex output,
00:16:25.08 and it is generated by 3 enzymes
00:16:27.28 in Module 4 of DEBS.
00:16:30.28 There is a ketoreductase,
00:16:34.03 a dehydratase,
00:16:36.04 and an enoylreductase,
00:16:38.08 that all collaborate with each other
00:16:41.06 to set this one stereocenter.
00:16:47.06 In addition to stereochemistry,
00:16:49.20 there is another very important
00:16:53.15 specificity that is encoded
00:16:56.11 in this assembly line
00:16:58.22 at each module,
00:17:00.24 and that is the specificity
00:17:05.17 that corresponds to the choice
00:17:08.19 of the extender unit.
00:17:10.19 In my introduction,
00:17:12.07 I pointed out that all of the modules
00:17:14.26 of 6-Deoxyerythronolide B synthase
00:17:18.04 use a methylmalonyl coenzyme A
00:17:21.14 extender unit.
00:17:23.22 In the case of DEBS,
00:17:27.06 the R group that is shown
00:17:29.25 in this enzymatic scheme
00:17:32.07 would be a methyl group.
00:17:34.17 Other polyketide synthases
00:17:37.06 can use coenzyme A thioesters
00:17:39.29 that contain other functional groups
00:17:42.16 in place of a methyl group, over here.
00:17:46.11 And all of these choices
00:17:48.18 are made by the acyltransferase.
00:17:53.00 These acyltransferases
00:17:55.10 are relatively specific.
00:17:58.01 Not only do they have high specificity,
00:18:01.04 as shown in this graph
00:18:03.15 for the coenzyme A precursor
00:18:05.28 they're picking from the metabolic soup...
00:18:09.09 so what you see in this graph over here
00:18:13.06 is the rate...
00:18:15.24 the velocity versus substrate concentration
00:18:18.20 of the preferred substrate,
00:18:20.20 which is methylmalonyl coenzyme A,
00:18:23.22 and down here are the rates
00:18:25.25 if R is one methyl short,
00:18:28.13 so in other words it's a hydrogen instead of a methyl,
00:18:31.14 or one methyl longer,
00:18:33.23 which is an ethyl group.
00:18:35.29 And as you can tell,
00:18:37.25 this acyltransferase
00:18:39.18 that we're showing you data for in this slide
00:18:42.08 is highly selective
00:18:44.24 for a methyl group
00:18:46.25 instead of one smaller or one larger.
00:18:50.16 Now, in addition to being specific
00:18:52.25 for its cognate substrate,
00:18:56.09 this enzyme is also specific
00:18:59.11 for its protein partner,
00:19:01.09 which is the acyl carrier protein,
00:19:03.22 that is being used.
00:19:05.25 And here I'm introducing you
00:19:07.25 to a concept that is gonna come back
00:19:10.11 in a more significant way
00:19:12.13 in the last of my three lectures,
00:19:14.16 which is the importance
00:19:16.10 of protein-protein interactions
00:19:19.04 in the assembly line biochemistry
00:19:22.00 of these systems.
00:19:23.22 In this case, what you're seeing is
00:19:27.03 that the acyl carrier protein
00:19:29.21 is being strongly recognized
00:19:32.00 by the acyltransferase,
00:19:34.05 because if you give this same acyltransferase
00:19:37.18 other acyl carrier proteins
00:19:39.25 from other modules of DEBS
00:19:41.23 or elsewhere,
00:19:43.19 they work much more poorly
00:19:46.09 than the natural acyl carrier protein.
00:19:49.19 So in addition to recognizing
00:19:51.15 the coenzyme A precursor,
00:19:53.27 you also have recognition
00:19:56.00 of the acyl carrier protein.
00:19:58.18 Now, for those of you who are familiar
00:20:00.16 with enzymes kinetics
00:20:02.16 know that from data like this,
00:20:04.18 you can derive mechanisms
00:20:06.10 of how these enzymes work.
00:20:08.15 So, in this case,
00:20:10.19 this acyltransferase
00:20:13.02 has a ping-pong
00:20:15.03 bi-bi-type of a mechanism.
00:20:18.11 The coenzyme A precursor first comes in,
00:20:21.21 it is bound by the acyltransferase,
00:20:25.05 the acyltransferase picks the methylmalonyl extender unit,
00:20:29.02 coenzyme A leaves,
00:20:30.23 the carrier protein comes in,
00:20:32.29 is recognized by the acyltransferase,
00:20:35.25 and takes away the product
00:20:38.06 - the methylmalonyl extender unit.
00:20:40.23 So that ping-pong element comes into
00:20:43.12 this kind of a mechanism.
00:20:45.12 Now, you can also ask,
00:20:46.29 in addition to these gatekeeper acyltransferases
00:20:50.25 that control the choice of the building block,
00:20:55.18 there are many other enzymes
00:20:57.18 in this assembly line
00:20:59.17 that lie downstream of each choice
00:21:02.15 that a module makes
00:21:04.16 of its building block.
00:21:06.09 To what extent do they influence
00:21:09.05 the overall substrate specificity?
00:21:11.29 Do they care about what the upstream module chose
00:21:16.17 as its precursor for elongating
00:21:20.01 the growing polyketide chain?
00:21:22.13 We can ask questions like that
00:21:24.16 using the assays... biochemical assays
00:21:27.11 I showed you earlier on,
00:21:29.16 and from those experiments you learn something
00:21:31.22 quite interesting.
00:21:33.16 So, you learn that the downstream steps,
00:21:36.14 beyond the acyltransfer step,
00:21:38.28 in many modules
00:21:41.12 are not that discriminatory,
00:21:43.09 analogous to what Henry Ford
00:21:45.11 had contemplated for his assembly line.
00:21:48.26 The downstream steps
00:21:51.01 have low, but not a significant amount,
00:21:54.16 of specificity.
00:21:56.21 So if, by whatever mechanism,
00:21:59.14 you can fool this acyltransferase
00:22:02.25 to put a hydrogen instead of a methyl
00:22:06.16 at this position on the carrier protein,
00:22:09.26 the elongation enzyme
00:22:12.06 that elongates the chain
00:22:14.10 and puts this R group in the growing polyketide chain,
00:22:19.14 primarily loses
00:22:22.12 about 2- to 4-fold specificity
00:22:25.10 as a result of this mistake
00:22:28.13 that the upstream step made.
00:22:31.00 That's not much in the grand scheme of things,
00:22:33.25 but what you have to remember is
00:22:37.02 these assembly lines have
00:22:39.18 many, many downstream steps.
00:22:41.27 So, one of these assembly lines,
00:22:43.23 the first module over here, or the second module,
00:22:46.28 has four or five modules downstream
00:22:50.17 that are looking at the consequences
00:22:53.01 of what that module did.
00:22:55.00 And so these small effects
00:22:57.09 at the level of substrate specificity
00:22:59.23 then have quite significant impact
00:23:02.21 on the final product,
00:23:04.18 and you can see this
00:23:06.18 in the context of assays like this.
00:23:09.06 So here,
00:23:10.24 in the spirit of engineering,
00:23:12.17 what we're doing is we're taking that natural...
00:23:15.17 the natural assembly line that nature uses
00:23:18.04 to make 6-Deoxyerythronolide B,
00:23:20.27 we're taking that full assembly line,
00:23:23.00 and instead of just presenting it
00:23:25.27 methylmalonyl coenzyme A,
00:23:28.14 we're now presenting it a 1:1 mixture
00:23:32.06 of methylmalonyl coenzyme A
00:23:34.11 and ethylmalonyl coenzyme A,
00:23:36.28 and we're asking what's gonna happen.
00:23:40.11 Are you gonna get just 6-Deoxyerythronolide B?
00:23:44.19 Are you gonna get something else,
00:23:47.10 one or two other products?
00:23:49.06 Or are you gonna get a zoo of products?
00:23:51.26 And the answer to that is,
00:23:53.28 you get some analogues
00:23:56.23 that are produced competitively
00:23:58.25 with the natural product 6-DEB,
00:24:02.23 but these compounds
00:24:05.24 aren't immediately obvious
00:24:08.00 why these should be formed
00:24:10.02 and other ones shouldn't be formed.
00:24:11.29 So at least one of these, for example,
00:24:13.27 this peak that I show you out here,
00:24:16.04 whose mass spectrum is shown over here,
00:24:18.22 we know with reasonable confidence,
00:24:21.06 has an ethyl group
00:24:23.07 that is incorporated at the C8 position
00:24:26.12 instead of a methyl group.
00:24:28.06 So, somehow,
00:24:30.10 that gatekeeper transferase
00:24:32.29 has enough tolerance
00:24:35.04 for an ethylmalonyl extender unit,
00:24:37.24 and all of the downstream enzymes
00:24:40.13 on the assembly line
00:24:42.11 are sufficiently tolerant
00:24:44.13 that they will let that mistake slide by,
00:24:46.28 so you get this desired product.
00:24:50.05 And if we could predict
00:24:52.10 what's gonna be made and what's not gonna be made
00:24:55.01 through an experiment like this,
00:24:57.05 why, then, we would have a way to precisely engineer
00:24:59.21 an antibiotic like erythromycin
00:25:01.21 to make a molecule like this.
00:25:03.27 But right now, we're just beginning to scratch
00:25:06.10 the tip of the iceberg
00:25:07.22 in terms of what's possible,
00:25:09.13 and what's not,
00:25:11.04 in these kinds of systems,
00:25:12.21 and an experiment like this illustrates
00:25:14.22 what's possible.
00:25:16.28 These kinds of insights can also be used
00:25:19.12 for most sophisticated engineering experiments,
00:25:22.07 analogous to the kinds of experiments
00:25:24.17 you may be familiar with
00:25:26.25 when you think about incorporating unnatural amino acids
00:25:31.18 in proteins that are derived
00:25:33.24 by ribosomal mechanisms.
00:25:36.04 And so, in this,
00:25:38.08 there are some examples of acyltransferases
00:25:42.10 that are what we call stand-alone acyltransferases.
00:25:46.12 They operate outside the assembly line,
00:25:49.21 and because they work
00:25:51.29 very, very fast compared to typical assembly line acyltransferases
00:25:58.03 that exist in assembly lines,
00:26:00.16 you can do some interesting experiments.
00:26:02.25 So in this case,
00:26:04.24 we have knocked out
00:26:07.10 one acyltransferase
00:26:09.17 out of all the six acyltransferases
00:26:12.09 on the 6-Deoxyerythronolide B synthase
00:26:16.23 -- so this is like a site-directed mutant
00:26:19.14 that has inactivated that acyltransferase --
00:26:23.02 and we complement it,
00:26:25.15 in trans,
00:26:27.26 with that very ultra-fast acyltransferase
00:26:30.27 that we get from a different assembly line
00:26:34.02 in nature.
00:26:36.07 And what happens is,
00:26:38.00 because this acyltransferase
00:26:40.09 will pick a malonyl unit instead of a methylmalonyl unit,
00:26:44.13 it can transfer that malonyl unit
00:26:47.07 onto this module,
00:26:49.02 because this module is simply waiting
00:26:50.26 for something to come its way,
00:26:52.29 and this enzyme can do it pretty quickly,
00:26:56.18 and now that resulting intermediate
00:26:58.27 moves all the way down in the assembly line
00:27:01.23 to give you a product
00:27:04.09 that has one and only one change
00:27:07.11 in the entire macrocycle
00:27:10.09 that is made.
00:27:12.08 And what you see over here is,
00:27:13.29 if I have my assembly line that is present
00:27:18.15 at say micromolar concentrations in my assay,
00:27:22.00 at nanomolar concentrations
00:27:24.26 I can get quite respectable incorporation
00:27:29.22 of malonyl coenzyme A
00:27:31.18 at that single position,
00:27:33.16 to give me 12-desmethyl-6-Deoxyerythronolide B.
00:27:39.25 So this is a way you can cheat the system,
00:27:43.26 so long as you have
00:27:46.19 a robust enzyme
00:27:49.00 that can trans-complement the module
00:27:51.15 that you wanna cheat.
00:27:53.22 So hopefully that gives you
00:27:55.24 a flavor of the kinds of tools we have
00:27:58.28 and the way we use these tools
00:28:01.01 to study specificity.
00:28:03.12 Thank you.

00:00:08.03 Hello again.
00:00:09.20 I'm Chaitan Khosla
00:00:11.11 from Stanford University,
00:00:13.10 and welcome to the third part
00:00:16.24 of this trilogy of lectures
00:00:18.29 on polyketide antibiotic biosynthesis
00:00:22.05 and assembly lines.
00:00:25.29 So, in the first lecture
00:00:27.21 I introduced you to the basics
00:00:30.02 of what an assembly line is,
00:00:33.13 what's the chemistry that happens on an assembly line,
00:00:37.03 and what an assembly line looks like.
00:00:41.15 We looked at the basic architecture
00:00:45.06 of the 6-Deoxyerythronolide B synthase,
00:00:48.19 or DEBS,
00:00:50.18 which I have used subsequently
00:00:52.27 to make most of the points
00:00:55.08 that I have made.
00:00:57.12 I introduced to you
00:00:59.16 the architecture of this assembly line,
00:01:01.25 or at least as best as we can gauge today
00:01:05.12 based on data we have.
00:01:07.24 In the second lecture
00:01:10.14 I introduced you to the tools,
00:01:13.09 and how we've used those tools
00:01:15.24 to study different kinds of questions.
00:01:18.23 So, we talked about refactoring
00:01:21.18 a metabolic pathway
00:01:23.24 like this assembly line of DEBS
00:01:26.03 in E. coli.
00:01:27.09 We talked about how to reconstitute that pathway
00:01:30.07 in a test tube.
00:01:32.02 I pointed out that you can
00:01:33.25 isolate individual reactions
00:01:36.12 on the assembly line
00:01:38.05 and look at the kinetics,
00:01:39.22 whether you're looking at extender unit selection,
00:01:41.24 translocation,
00:01:43.08 elongation,
00:01:44.27 modification events.
00:01:46.24 We also talked about how these tools
00:01:48.23 can be used to study specificity
00:01:51.03 and, in particular,
00:01:52.29 we talked about two types of specificities:
00:01:56.00 stereospecificity,
00:01:58.24 I mentioned that stereochemistry
00:02:01.06 is controlled by reductive domains,
00:02:03.23 as well as extender unit specificity,
00:02:07.17 and I mentioned, over there,
00:02:09.25 you not only have enzyme-substrate recognition
00:02:13.15 but also protein-protein recognition.
00:02:18.04 This final lecture focuses on what, in my mind,
00:02:22.10 is the grandest challenge of all
00:02:25.03 in this field:
00:02:27.05 understanding how an assembly line
00:02:30.17 actually comes about.
00:02:33.20 What do I mean by that?
00:02:37.05 I've been showing you,
00:02:39.06 quite freely I might add,
00:02:41.00 schemes like this
00:02:43.08 throughout my earlier two lectures.
00:02:47.20 Every time I show you one of these schemes
00:02:50.08 or, for that matter,
00:02:52.04 you could open your favorite journal of biochemistry today,
00:02:55.26 and chances are in any given issue,
00:02:58.07 you'll find somebody having published
00:03:00.14 yet another one of these
00:03:02.13 kinds of assembly lines
00:03:04.04 that they have cloned, sequenced,
00:03:05.21 and started to characterize.
00:03:07.07 And in all cases you'll find
00:03:09.01 cartoons like the kind you see in this scheme.
00:03:15.26 The question that lies ahead of us is,
00:03:18.11 how do we know that
00:03:21.02 this is how the assembly line works?
00:03:26.27 And one way to recognize
00:03:29.12 why that question
00:03:31.25 is not an obvious question
00:03:34.15 is by thinking back
00:03:36.08 to all those orphan polyketide assembly lines
00:03:39.23 that I talked about to you
00:03:41.27 at the beginning of my first lecture.
00:03:43.29 So, I mentioned to you,
00:03:45.29 there's these large numbers of orphan assembly lines
00:03:49.03 that make polyketides
00:03:51.10 whose structure we don't know.
00:03:54.14 If I gave you the sequence
00:03:57.02 of one of those assembly lines
00:03:59.17 and asked you
00:04:01.17 to draw me an equivalent scheme
00:04:03.06 like the kind you see in front of you
00:04:05.02 in this slide,
00:04:07.10 chances are you won't be able to do it
00:04:09.15 with any confidence.
00:04:11.26 And the reason why I can show
00:04:15.07 this scheme in this slide,
00:04:16.24 and hold my head up high,
00:04:19.03 is because I know the answer
00:04:23.07 for this assembly line.
00:04:25.26 I know the structure
00:04:27.20 of 6-Deoxyerythronolide B,
00:04:30.13 and using that knowledge I am essentially
00:04:34.14 back-programming on this assembly line
00:04:37.08 what must happen at each stage
00:04:40.07 in order to get that final product.
00:04:43.20 But that begs the question,
00:04:45.27 how does nature know
00:04:48.01 that this is how the assembly line must work?
00:04:50.27 Why does nature start here,
00:04:54.01 and move here?
00:04:55.29 Or putting it differently,
00:04:57.22 why can't nature take a product
00:05:00.09 from this module
00:05:02.09 and instead of passing it on to this module,
00:05:05.12 why can't it just skip these modules altogether
00:05:08.21 and pass it on to this?
00:05:10.29 Or, why can't it take this precursor
00:05:15.13 and pass it to some other polyketide synthase
00:05:18.05 that's floating around in the same cell?
00:05:21.20 Where are the rules
00:05:24.17 that dictate how this vectorial translocation
00:05:28.17 of polyketides
00:05:30.15 must occur in the manner
00:05:32.11 that I tell you it's occurring
00:05:34.20 in this scheme?
00:05:36.20 That is the fundamental challenge
00:05:39.21 associated with this field.
00:05:43.27 The way we think about this challenge
00:05:46.23 goes back to the work
00:05:50.08 of an inspirational enzymologist
00:05:54.26 from a previous generation,
00:05:56.16 whose name you may have heard:
00:05:58.25 Bill Jencks, or William P. Jencks.
00:06:02.29 Jencks made huge contributions
00:06:06.07 to our understanding
00:06:08.08 of how enzymes work.
00:06:10.15 One of the problems he talked about
00:06:12.29 and gave a lot of thought to,
00:06:15.09 as is discussed in this article
00:06:17.07 that I would encourage any student
00:06:19.08 who's interested in machines in biology
00:06:24.03 to read,
00:06:25.21 was the question of how do you get
00:06:28.24 vectorial processes occurring in nature,
00:06:32.19 off the back of proteins like enzymes?
00:06:37.13 This article is chock-full
00:06:40.05 of many really interesting insights
00:06:42.21 that were way ahead of their time.
00:06:46.01 I certainly don't have the time
00:06:47.27 to go into all of them.
00:06:50.03 But there's one insight
00:06:51.27 that I'm going to focus on
00:06:53.21 because I think it does a very nice job
00:06:55.27 of framing the understanding
00:06:58.11 of the vectorial chemistry
00:07:00.05 that I'm here to tell you about.
00:07:02.26 What Jencks says in this article
00:07:07.03 is that proteins that do coupled vectorial processes,
00:07:10.27 that do vectorial processes,
00:07:13.06 differ from your garden variety enzyme
00:07:16.13 in the sense that they undergo large changes
00:07:21.08 in substrate specificity
00:07:23.14 at different points in their overall catalytic cycle.
00:07:29.26 And a really good example
00:07:32.14 that he uses in his quintessential
00:07:35.01 chicken-scratch cartoon manner
00:07:37.27 that's shown over here
00:07:40.08 is the example of how
00:07:43.03 myosin moves on actin fibers.
00:07:46.15 That you probably know about
00:07:49.00 because it's what textbooks are made up...
00:07:51.09 biochemistry textbooks are made of.
00:07:53.18 What you know is that myosin
00:07:56.07 moves along an actin highway
00:07:59.16 that is made up of actin monomers
00:08:02.15 by essentially going from one monomer of actin
00:08:07.15 to the next and so on.
00:08:11.07 And that movement
00:08:13.18 is powered by the hydrolysis of ATP,
00:08:17.19 by myosin.
00:08:19.28 And what Jencks postulated back then,
00:08:22.14 or what he articulated in this paper,
00:08:25.28 was that the reason you get this vectorial movement
00:08:32.07 is because there are different states of myosin
00:08:35.15 at different points in the overall cycle,
00:08:39.15 and some of these states
00:08:42.07 bind tightly to myosin,
00:08:44.22 the unbound states,
00:08:47.03 whereas ATP- or ADP-bound states,
00:08:50.19 the myosin does not bind very tightly
00:08:54.00 to actin.
00:08:56.21 The key point that Jencks made
00:08:59.14 is the reason this is a machine,
00:09:01.22 and it's not like myosin
00:09:03.23 dances back and forth on this actin highway,
00:09:07.09 the reason it motors along this actin highway
00:09:10.25 is because there is a distinct change
00:09:14.27 in specificity of myosin for actin
00:09:18.14 at different points in the catalytic cycle.
00:09:21.21 When myosin binds to ATP,
00:09:26.01 it exchanges with an unbound state
00:09:32.03 that binds to one monomer of actin,
00:09:36.02 whereas when that ATP is hydrolyzed,
00:09:39.23 that myosin sets up the binding
00:09:44.09 of a distinct conformational state
00:09:46.29 to the next.
00:09:49.03 It can't...
00:09:51.11 this exchange between the bound and the unbound state
00:09:54.29 is prohibited, because if this happened
00:09:57.03 you would be wasting this ATP hydrolysis,
00:10:00.21 and you'd be back to where you started from.
00:10:04.01 But the reason this moves forward
00:10:06.20 is because this state
00:10:09.00 has a distinct specificity
00:10:12.23 than this state of the enzyme.
00:10:15.27 So the big changes
00:10:19.02 in the specificity of myosin
00:10:21.13 are what Jencks proposed
00:10:23.12 is the basis for this vectorial process.
00:10:28.02 We think about our challenge
00:10:32.06 very much analogous
00:10:34.17 to how Jencks proposed
00:10:37.13 how myosin works on actin.
00:10:39.29 And more specifically,
00:10:42.10 there are two postulates
00:10:44.20 that we draw upon
00:10:46.28 from Jencks' analogy
00:10:49.05 to actin and myosin
00:10:51.07 to explain how this vectorial process happens.
00:10:55.08 The first one
00:10:57.19 is that the specificity change
00:11:00.14 that Jencks talked about,
00:11:03.05 in our context,
00:11:05.23 happens at the level of protein-protein interactions.
00:11:09.26 I gave you an example
00:11:12.04 in my previous lecture
00:11:14.04 of an acyltransferase
00:11:16.21 that has protein-protein specificity
00:11:19.14 in addition to its specificity
00:11:21.24 for its normal substrate.
00:11:23.18 I'll be elaborating on this theme
00:11:25.19 much more in this lecture.
00:11:28.07 And the second hypothesis
00:11:31.09 that helps us think about assembly line
00:11:34.20 vectorial biochemistry
00:11:37.03 is that this core of the protein
00:11:39.22 that exists in each module,
00:11:41.20 this KS and AT,
00:11:44.26 has specificity for the acyl carrier protein,
00:11:50.15 in an interesting way,
00:11:52.20 that leads to establishing directionality.
00:11:55.26 Bear with me if that's not clear.
00:11:58.10 Let me first start by giving you
00:12:00.14 some information, some background
00:12:02.18 on the relevance of protein-protein interactions.
00:12:05.17 So, about 15 years ago,
00:12:09.07 when Rajesh Gokhale was in my lab,
00:12:12.08 he came up with an interesting postulate
00:12:15.27 about how assembly line directionality happens.
00:12:21.02 He hypothesized
00:12:22.27 that specificity that leads to this chain
00:12:26.07 moving from this module to this module
00:12:29.08 is not in the active sites
00:12:32.12 for different substrates,
00:12:35.04 but lies at the interface
00:12:37.18 between these modules.
00:12:39.22 So you have these docking sites
00:12:43.08 that exist between modules,
00:12:46.02 that are orthogonal
00:12:48.13 to the core modules
00:12:50.22 that are doing the actual enzymology,
00:12:53.19 that allow two modules
00:12:56.05 to come together
00:12:58.04 so that you have this directionality
00:13:01.05 that is established.
00:13:03.03 How do we know that?
00:13:04.28 We know that through experiments like this.
00:13:07.19 This is one example of an experiment
00:13:09.14 that Stuart Tsuji did in my lab
00:13:11.26 some years ago
00:13:14.08 where he kinetically isolated,
00:13:16.25 from the entire DEBS assembly line
00:13:19.28 you see over here,
00:13:22.03 these two modules,
00:13:24.00 Module 2 and Module 3.
00:13:25.21 He pulled them out of the overall system,
00:13:28.09 put them into a test tube,
00:13:30.09 and reconstituted their full enzymology
00:13:33.16 that's shown in this scheme,
00:13:36.06 measuring the appropriate parameters
00:13:38.21 that one gets from these kinds of experiments.
00:13:43.05 Now, what he does is
00:13:45.19 he switches these short peptide linkers
00:13:48.11 that flank these modules
00:13:50.25 for their counterparts from this module.
00:13:54.29 And if he switches just one of the two linkers,
00:13:58.25 what he gets are two modules
00:14:01.18 that by themselves
00:14:03.25 are just as good of catalysts as they were here,
00:14:08.02 but when you put them together,
00:14:10.04 they are simply unable to talk to each other.
00:14:15.07 If, however,
00:14:16.26 you take this module that has this linker
00:14:19.08 instead of this linker,
00:14:21.25 and couple it to this module
00:14:26.23 that has this linker
00:14:28.29 instead of this linker,
00:14:31.12 this pair over here
00:14:33.18 is indistinguishable from this pair.
00:14:36.23 And so now you see
00:14:39.06 how these matching docking sites
00:14:43.22 provide for the ability for Module 2
00:14:47.02 to talk to Module 3,
00:14:49.20 as opposed to Module 5
00:14:51.23 or any of the other modules.
00:14:53.25 So you're seeing the establishing of directionality
00:14:57.14 off the back of protein-protein interactions.
00:15:02.27 Now, what the Gokhale/Tsuji experiments do
00:15:05.18 is they tell us why Module talks to Module 3.
00:15:09.21 What they don't tell us is,
00:15:12.01 why don't chains go back and forth?
00:15:15.00 It's analogous to the question
00:15:17.09 that Jencks was asking:
00:15:18.28 why is it that myosin moves
00:15:20.21 in only one direction on actin,
00:15:23.12 as opposed to just going back and forth,
00:15:25.19 back and forth?
00:15:27.20 That question has an exact analogy over here,
00:15:30.27 because as you'll remember from the first lecture
00:15:33.11 of my three lectures,
00:15:35.06 I introduced to you that chain translocation
00:15:38.09 -- when a chain moves from one module
00:15:40.02 to the next --
00:15:41.24 it does so by a thiol
00:15:43.27 to a thioester exchange reaction.
00:15:47.05 And that reaction
00:15:48.26 can just as easily run in the backward direction
00:15:51.03 as it can run in the forward direction.
00:15:53.23 And so what precludes chains
00:15:55.22 from just going back and forth
00:15:57.15 and messing up the chemistry
00:15:59.06 so you'll never get an antibiotic
00:16:00.24 out of these assembly lines?
00:16:02.22 What's written in this code
00:16:04.17 that allows that to happen?
00:16:09.05 So, the question is,
00:16:12.06 how does this event
00:16:14.25 lead to a movement here,
00:16:17.01 but when it comes time to move the chain forward,
00:16:20.14 it doesn't just go back
00:16:22.15 into the same module,
00:16:24.09 but rather goes to the next module.
00:16:29.12 Again, over here,
00:16:31.17 protein-protein interactions
00:16:33.19 play a very important role.
00:16:35.13 And in particular,
00:16:37.24 the interactions,
00:16:40.04 not just in these docking sites
00:16:42.00 that Gokhale and Tsuji identified,
00:16:43.29 but also between this protein
00:16:46.22 and this protein,
00:16:48.21 play a very critical role.
00:16:50.23 How do we know that?
00:16:52.17 Through experiments like this
00:16:54.11 that Nick Wu did,
00:16:56.06 where he isolated, kinetically,
00:16:58.02 this chain translocation event,
00:17:01.26 exposed this module over here
00:17:06.08 to the same substrate,
00:17:08.07 same docking partners,
00:17:10.15 same receiving module,
00:17:12.22 but now he changes
00:17:14.19 the identity of the donor carrier protein.
00:17:17.20 So all that's being changed over here
00:17:19.19 is the identity of this protein.
00:17:22.05 And what you see is
00:17:24.11 you have a big difference
00:17:26.19 in specificity constants
00:17:28.24 just based upon who this player is
00:17:32.19 in the overall chain translocation event,
00:17:35.11 suggesting that these protein-protein interactions
00:17:39.08 are very important.
00:17:41.17 We now know,
00:17:43.06 through work of Shiven Kapur
00:17:45.07 and others in my lab,
00:17:47.01 what the basis for this is.
00:17:49.11 So, it turns out that these carrier proteins,
00:17:51.29 which are helical proteins,
00:17:54.11 have different helices
00:17:56.10 -- different portions of this helical protein --
00:17:59.12 that dictate specificity at different events.
00:18:02.22 How do we know that?
00:18:05.02 So, you can take this red carrier protein
00:18:07.23 that talks to this red acceptor module,
00:18:12.06 replace it with the green carrier protein,
00:18:15.05 it doesn't do a good job of talking
00:18:17.14 to this acceptor module,
00:18:19.12 but now if you just put a portion of this helix
00:18:22.14 into this protein,
00:18:24.14 now this chimeric protein
00:18:26.19 is just as good,
00:18:28.12 or almost just as good,
00:18:30.06 as this red carrier protein
00:18:32.23 in delivering cargo
00:18:34.21 through this chain translocation event,
00:18:37.10 suggesting that it's this helix over here
00:18:41.02 that plays a very important role
00:18:43.08 in dictating the specificity
00:18:45.02 during chain translocation.
00:18:47.28 You can do exactly analogous experiments
00:18:50.05 in that other theoretical reaction,
00:18:53.22 the chain elongation reaction
00:18:55.23 where that carbon-carbon bond is formed
00:18:58.00 between the extender unit and the growing polyketide chain
00:19:01.23 to give you this newly elongated chain.
00:19:04.25 You can kinetically isolate this event,
00:19:07.10 you can use the same substrates,
00:19:10.20 same catalysts,
00:19:12.28 just change the identity of this carrier protein.
00:19:16.06 And what you see is,
00:19:17.28 depending on which module you use,
00:19:19.24 you can have big effects
00:19:21.23 on the specificity constant over here,
00:19:24.11 as Alice Chen showed in these experiments.
00:19:27.22 You can use the same kind of approaches
00:19:30.10 that Shiven Kapur used
00:19:32.05 to make chimeric carrier proteins
00:19:34.11 and show which portions
00:19:36.16 of these carrier proteins
00:19:38.12 play a critical role in dictating the specificity
00:19:42.22 that is measured in these parameters.
00:19:45.11 And so the take-home message
00:19:47.23 from experiments like this
00:19:51.03 is that even though chain elongation
00:19:55.07 and chain translocation
00:19:57.19 are sequential events
00:20:00.19 along the assembly line,
00:20:02.16 and even though all of those events
00:20:04.24 involve ketosynthase-acyl carrier protein interactions,
00:20:09.14 the specificity of those events
00:20:12.04 is being driven by different faces
00:20:14.23 at this carrier protein.
00:20:17.21 So this blue face of this carrier protein
00:20:20.26 is what's driving the translocation reaction,
00:20:24.17 whereas the yellow face of the carrier protein
00:20:27.19 is what's driving the elongation reaction.
00:20:31.20 So you have this Janus-like biochemistry
00:20:36.01 in these carrier proteins.
00:20:38.19 And the reason that allows you to do things
00:20:42.19 in a vectorial manner
00:20:45.13 is because now you can set up
00:20:48.18 so that a carrier protein
00:20:51.06 can only recognize the next module.
00:20:54.06 How do we know that?
00:20:56.01 We know that through experiments
00:20:57.23 where we engineer these carrier proteins
00:21:00.06 to be confused carrier proteins.
00:21:03.07 This is one example of that experiment,
00:21:05.16 where we engineer a module
00:21:07.29 that stutters.
00:21:10.09 So if you take a natural module,
00:21:12.12 like this one shown over here,
00:21:14.09 and its own carrier protein over here,
00:21:16.18 you give it a nucleophilic substrate
00:21:18.23 and electrophilic growing polyketide chain,
00:21:22.10 and you ask it to catalyze chain elongation,
00:21:25.00 it'll do so,
00:21:27.06 it'll do one round of chain elongation,
00:21:31.01 and it'll stop at this point.
00:21:34.07 And the reason it stops at this point
00:21:36.28 and doesn't go back
00:21:39.09 to the same carrier protein
00:21:41.08 is that this carrier protein
00:21:43.27 is a partner of this ketosynthase
00:21:46.17 in this chain elongation reaction
00:21:49.10 that forms this critical carbon-carbon bond,
00:21:52.22 but it is not a partner
00:21:55.16 for chain translocation.
00:21:57.16 Its chain translocation partner
00:21:59.13 is elsewhere on that assembly line.
00:22:02.14 And so the way you can test that
00:22:05.22 is you can take the recognition features
00:22:08.14 of the next carrier protein,
00:22:11.29 build them into this system
00:22:14.16 to generate a chimeric carrier protein
00:22:17.26 that can not only do a chain elongation event,
00:22:21.13 but this chimeric thing
00:22:23.12 can then donate it back
00:22:25.10 to this ketosynthase
00:22:27.19 and go through yet another round of chain elongation
00:22:30.11 to give you a longer module.
00:22:32.15 So here you've got a module
00:22:34.19 that is stuttering;
00:22:37.18 that's doing two rounds of elongation
00:22:40.07 instead of one and only one round,
00:22:42.10 because we've kind of scrambled up its recognition features.
00:22:47.02 And so what that allows me to do
00:22:49.10 is to leave you with a basic picture
00:22:52.17 of how you might wanna think about this assembly line.
00:22:56.12 So, I showed you this cartoon.
00:22:59.27 The question we're asking is,
00:23:01.29 how does the growing polyketide chain
00:23:05.10 pick one and only one path?
00:23:07.17 It's going through at least
00:23:09.17 300 Ångstroms of distance
00:23:11.19 as it travels through these modules
00:23:14.21 from one active site to another,
00:23:17.08 and comes out as a 6-Deoxyerythronolide B.
00:23:21.11 How does it pick one and only one path
00:23:24.08 to take through this entire assembly line?
00:23:27.27 And so, if we isolate just these two modules,
00:23:31.05 Modules 2 and 3,
00:23:33.24 I can show you what I think
00:23:36.05 might be a model for how this works.
00:23:39.18 So we start off
00:23:42.00 with the upstream module,
00:23:44.12 this pink module,
00:23:46.26 bound to its carrier protein
00:23:49.16 in the elongation state
00:23:51.19 -- what Jencks would call...
00:23:53.28 has specificity for the elongation state --
00:23:57.06 and that specificity
00:23:59.06 is derived from this yellow portion
00:24:01.07 of the carrier protein
00:24:03.11 that docks into this active site
00:24:05.14 and puts its substrate
00:24:07.17 where it belongs.
00:24:09.25 Meanwhile, the green carrier protein
00:24:11.24 of this green module
00:24:13.28 is just sitting in neutral mode.
00:24:16.25 So this carrier protein
00:24:19.02 then catalyzes chain elongation,
00:24:22.26 it yanks out that chain,
00:24:25.01 it can't return it back to the same site
00:24:27.05 because it doesn't have the appropriate recognition features,
00:24:30.27 instead its got recognition features, now,
00:24:34.00 using that panhandle linker,
00:24:36.06 that docking site that was identified as well as this helix
00:24:41.14 that Kapur identified.
00:24:44.02 It docks into this module,
00:24:46.11 but in a different orientation,
00:24:48.11 so now it can translocate
00:24:50.20 the chain to the next module.
00:24:54.14 When that happens,
00:24:56.13 that carrier protein goes back
00:24:58.13 into the neutral mode
00:25:00.15 and this carrier protein,
00:25:02.03 which is charged with the extender unit,
00:25:04.09 comes in to do the elongation reaction.
00:25:07.15 And so this three-stroke process
00:25:10.11 goes through again and again
00:25:12.19 to give you the final product,
00:25:15.19 to allow chains
00:25:17.13 to move forward through the assembly line.
00:25:21.10 And so I'll leave you with this picture.
00:25:24.19 I started with the picture to the left,
00:25:27.13 to motivate these remarkable antibiotic machines.
00:25:31.16 I've added one more picture
00:25:33.11 that I suspect most of you
00:25:34.26 who've had a class in biochemistry recognize.
00:25:38.10 It is the picture that we often use
00:25:40.18 to describe how proteins are made
00:25:43.06 on ribosomes.
00:25:47.24 Let me ask you the following question:
00:25:51.03 what do these pictures mean to you?
00:25:54.27 Chances are if I just flashed
00:25:57.11 both these pictures in front of you,
00:26:00.10 you would immediately recognize
00:26:02.11 what I'm saying.
00:26:04.27 If, on the other hand,
00:26:07.08 you had just arrived today from Mars,
00:26:10.08 and I showed you these pictures,
00:26:13.02 they would mean absolutely nothing to you.
00:26:16.29 So why do these pictures mean so much to you?
00:26:21.00 I would submit to you they mean a lot to you
00:26:24.03 because you have seen the movie in each case,
00:26:28.04 and that is where I will leave you
00:26:30.21 as the future of my field.
00:26:35.12 What I showed you in the past three slides
00:26:39.13 were essentially the first three frames
00:26:42.22 of a movie
00:26:45.15 that will be the DEBS movie
00:26:47.26 over the next decade.
00:26:50.09 There's no Hollywood,
00:26:51.26 no Disney as yet,
00:26:54.06 but it's backed up by
00:26:58.01 data based upon...
00:27:00.11 through a combination of biochemistry,
00:27:03.14 structural biology,
00:27:05.09 and similar types of chemical/biological approaches.
00:27:10.01 Our challenge for this field
00:27:11.27 is to take those static pictures
00:27:15.06 that I've been showing you so far
00:27:17.15 and convert them into a movie,
00:27:19.22 so once you've seen that movie,
00:27:21.29 you will recognize the engineering implications
00:27:25.01 of every state
00:27:27.07 in every one of those thousands of assembly lines
00:27:30.20 that nature has give you,
00:27:32.21 and be able to rationally think
00:27:34.23 about how to recombine them,
00:27:36.23 or how to extract interesting antibiotics
00:27:39.23 from the right kind of assembly line in nature.
00:27:43.06 I can't do that today,
00:27:46.17 nor can any of my coworkers.
00:27:49.24 But I hope this inspires you
00:27:52.16 to think about how that might be done in the future.
00:27:55.03 This brings me to the end of the lecture.
00:27:58.16 I'd like to thank you for your attention.
00:28:02.24 I'd like to thank all of my students
00:28:05.16 and postdoctoral fellows
00:28:07.21 who, over the years,
00:28:09.16 have worked with me on DEBS
00:28:11.26 and related polyketide assembly lines.
00:28:14.29 Their names show up to the left of the slide,
00:28:18.27 and I've been acknowledging their papers
00:28:21.18 as we've gone along through
00:28:23.20 these different modules.
00:28:26.27 And I'd like to also thank my collaborators:
00:28:30.20 David Cane at Brown University,
00:28:33.05 with whom I've had a long-standing collaboration
00:28:36.19 on the study of DEBS,
00:28:38.23 as well as my collaborators
00:28:41.00 at the synchrotron
00:28:42.23 -- SLAC synchrotron --
00:28:44.21 Matthews Irimpan,
00:28:46.02 Tsutomu Matsui,
00:28:47.25 and Thomas Weiss,
00:28:49.11 with whom we've had very productive collaborations
00:28:51.25 associated with the
00:28:54.05 crystallographic and SAXS characterization
00:28:56.29 of DEBS.
00:28:58.12 And last, but not least,
00:29:00.03 I'd like to thank the NIH
00:29:02.01 for having supported all our work on DEBS
00:29:05.13 over the past 15 years.
00:29:07.27 Thank you.