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Session 4: An Introduction to Polyketide Assembly Lines

Transcript of Part 1: 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.

This material is based upon work supported by the National Science Foundation and the National Institute of General Medical Sciences under Grant No. MCB-1052331. Any opinion, finding, conclusion, or recommendation expressed in these videos are solely those of the speaker and do not necessarily represent the views of iBiology, the National Science Foundation, the National Institutes of Health, or other iBiology funders.

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