WEBVTT 00:00:00.500 --> 00:00:02.810 The following content is provided under a Creative 00:00:02.810 --> 00:00:04.380 Commons license. 00:00:04.380 --> 00:00:06.670 Your support will help MIT OpenCourseWare 00:00:06.670 --> 00:00:11.010 continue to offer high quality educational resources for free. 00:00:11.010 --> 00:00:13.670 To make a donation or view additional materials 00:00:13.670 --> 00:00:17.600 from hundreds of MIT courses, visit MIT OpenCourseWare 00:00:17.600 --> 00:00:18.460 at ocw.mit.edu. 00:00:25.494 --> 00:00:27.760 ELIZABETH NOLAN: --by talking about ClpX. 00:00:27.760 --> 00:00:31.720 And then we're going to move into module 4-- 00:00:31.720 --> 00:00:34.930 which is the last module before spring break-- 00:00:34.930 --> 00:00:38.800 on synthases and assembly line biosynthesis. 00:00:44.690 --> 00:00:51.640 So basically last time, where we left off is, 00:00:51.640 --> 00:00:53.560 we went over experiments that were 00:00:53.560 --> 00:00:56.920 done to look at denaturation, translocation, 00:00:56.920 --> 00:00:59.910 and degradation by ClpXP. 00:00:59.910 --> 00:01:04.390 And closed with a question about what actually is going on 00:01:04.390 --> 00:01:07.930 in ClpX with this ATP binding and hydrolysis 00:01:07.930 --> 00:01:11.350 to allow for these condemned protein 00:01:11.350 --> 00:01:14.410 substrates to be unfolded and translocated 00:01:14.410 --> 00:01:16.900 into the degradation chamber. 00:01:16.900 --> 00:01:23.140 And I left you just with the statement that although we 00:01:23.140 --> 00:01:37.800 think about ClpX as this hexamer that has six identical 00:01:37.800 --> 00:01:41.160 subunits, what studies have shown is that there's some 00:01:41.160 --> 00:01:44.988 inherent asymmetry within this AAA+ ATPase. 00:01:44.988 --> 00:01:47.280 And that's what we're going to talk about a little bit. 00:01:47.280 --> 00:01:50.550 So this is just a slide from a few lectures ago 00:01:50.550 --> 00:01:53.880 that's showing the top view and side view of ClpX 00:01:53.880 --> 00:01:55.260 and how we've thought about this. 00:01:55.260 --> 00:01:58.470 And I think these studies just really 00:01:58.470 --> 00:02:02.940 highlight how complicated these machines are 00:02:02.940 --> 00:02:05.100 and that there's still a lot more we 00:02:05.100 --> 00:02:06.780 need to figure out here. 00:02:06.780 --> 00:02:10.860 So as I said last time, this asymmetry 00:02:10.860 --> 00:02:15.330 comes from whether or not each ClpX subunit 00:02:15.330 --> 00:02:17.460 is bound to nucleotide. 00:02:17.460 --> 00:02:22.440 And so basically, from looking at many different crystal 00:02:22.440 --> 00:02:35.410 structures, what can be done is that the ClpX subunits 00:02:35.410 --> 00:02:43.960 can be divided into two different types based 00:02:43.960 --> 00:02:51.950 on conformation here. 00:02:51.950 --> 00:02:53.570 And so in thinking about this, we 00:02:53.570 --> 00:02:58.730 want to first think about the ClpX domain organization. 00:02:58.730 --> 00:03:02.240 And if we just think about this, what ClpX has is 00:03:02.240 --> 00:03:05.930 an end domain followed by a domain 00:03:05.930 --> 00:03:11.660 that's called the large domain and then followed 00:03:11.660 --> 00:03:12.905 by a small domain. 00:03:18.050 --> 00:03:22.520 So 633 amino acids, just to give you a sense of size, 00:03:22.520 --> 00:03:27.430 and about 69 kilodaltons per subunit. 00:03:27.430 --> 00:03:29.180 And so what we're going to focus on 00:03:29.180 --> 00:03:31.880 are the large and the small subunits 00:03:31.880 --> 00:03:35.930 and what's observed from many different crystal structures. 00:03:35.930 --> 00:03:40.130 And so these two different types of subunit 00:03:40.130 --> 00:03:43.940 have been described as loadable and unloadable, 00:03:43.940 --> 00:03:48.100 and that depends on whether or not nucleotide is bound. 00:03:48.100 --> 00:03:51.890 So if we consider of these two types, 00:03:51.890 --> 00:03:55.040 just thinking about the large and small domains, 00:03:55.040 --> 00:04:01.000 we have this loadable arrangement 00:04:01.000 --> 00:04:02.470 which binds nucleotide. 00:04:08.340 --> 00:04:11.245 And in cartoon, something like this. 00:04:11.245 --> 00:04:15.626 So we have the large subunit. 00:04:15.626 --> 00:04:16.709 We have the small subunit. 00:04:20.310 --> 00:04:22.304 And we have this region that's called a hinge. 00:04:26.520 --> 00:04:28.260 So this is one ClpX. 00:04:33.915 --> 00:04:35.060 So ATP binds. 00:04:38.140 --> 00:04:42.080 And so the other type is described as unloadable. 00:04:48.390 --> 00:04:54.060 And this type of subunit does not bind 00:04:54.060 --> 00:04:57.090 nucleotide when in this unloadable conformation. 00:05:00.760 --> 00:05:03.495 And so we can draw this. 00:05:03.495 --> 00:05:05.120 Here, again, we have the large subunit. 00:05:08.430 --> 00:05:11.040 And there's a change in conformation. 00:05:11.040 --> 00:05:13.740 And here's the small subunit here. 00:05:16.320 --> 00:05:20.970 So what's been found from looking at many crystal 00:05:20.970 --> 00:05:26.580 structures is that within the ClpX hexamer, 00:05:26.580 --> 00:05:29.820 there's an arrangement of these loadable and unloadable 00:05:29.820 --> 00:05:31.140 subunits. 00:05:31.140 --> 00:05:40.960 So in many crystals, what's found 00:05:40.960 --> 00:05:44.018 is that there's four loadable-- 00:05:44.018 --> 00:05:45.310 I'm just going to do with "L"-- 00:05:45.310 --> 00:05:55.240 plus two unloadable subunits arranged with about 00:05:55.240 --> 00:05:57.040 two-fold symmetry, so LULLUL. 00:06:01.960 --> 00:06:04.380 So there's some asymmetry in the subunits. 00:06:12.640 --> 00:06:15.130 And so also from these crystal structures, 00:06:15.130 --> 00:06:16.840 there's some more observations that we 00:06:16.840 --> 00:06:20.380 don't see with just these cartoons of the 6-mer. 00:06:20.380 --> 00:06:24.190 So we can learn about how subunits interact, of course, 00:06:24.190 --> 00:06:25.975 and this is what's shown. 00:06:42.230 --> 00:06:44.480 So if we look at these structures 00:06:44.480 --> 00:06:48.200 and consider how these subunits interact, what we find 00:06:48.200 --> 00:06:52.060 is that the small subunit of one ClpX-- 00:06:52.060 --> 00:06:56.630 or sorry, the small domain of one ClpX subunit interacts 00:06:56.630 --> 00:07:01.470 with the large domain of the adjacent ClpX subunit. 00:07:01.470 --> 00:07:02.645 And so we can draw this. 00:07:06.050 --> 00:07:12.030 Basically, if we consider a large domain-- 00:07:12.030 --> 00:07:13.880 and let's say this is subunit 2-- 00:07:17.000 --> 00:07:20.160 then what we find is that there's 00:07:20.160 --> 00:07:27.890 the small domain and then the large domain 00:07:27.890 --> 00:07:29.540 of subunit next door. 00:07:29.540 --> 00:07:31.540 So let's call this subunit 1. 00:07:34.340 --> 00:07:40.010 So here's our hinge of subunit 1, 00:07:40.010 --> 00:07:44.150 and ATP binding happens in here. 00:07:44.150 --> 00:07:46.700 So we can think about this arrangement. 00:07:46.700 --> 00:07:53.210 And then what's been defined is something called a rigid body. 00:07:53.210 --> 00:07:56.690 And so this rigid body is comprised 00:07:56.690 --> 00:08:00.590 of the large domain of one subunit 00:08:00.590 --> 00:08:03.800 and the small domain of the next. 00:08:07.400 --> 00:08:08.240 Rigid body. 00:08:23.620 --> 00:08:40.663 So large domain of one ClpX and small domain of another subunit 00:08:40.663 --> 00:08:41.330 that's adjacent. 00:08:49.710 --> 00:08:55.430 So in thinking about this, we can consider the ClpX hexamer 00:08:55.430 --> 00:08:56.960 in another way. 00:08:56.960 --> 00:08:59.270 So how I've initially presented it to you 00:08:59.270 --> 00:09:04.250 when we introduced these oligomers is just as a 6-mer, 00:09:04.250 --> 00:09:04.750 right? 00:09:04.750 --> 00:09:06.110 6 subunits. 00:09:06.110 --> 00:09:08.750 But another way to think about ClpX 00:09:08.750 --> 00:09:14.630 is that it's actually six rigid bodies that 00:09:14.630 --> 00:09:18.560 are connected by hinges, where each rigid body has 00:09:18.560 --> 00:09:22.760 a component from two subunits, a large domain from one 00:09:22.760 --> 00:09:23.840 and a small from another. 00:09:26.720 --> 00:09:32.000 And so the hinges are within a single subunit 00:09:32.000 --> 00:09:35.690 based on this cartoon where ATP binds. 00:09:35.690 --> 00:09:40.310 And so the thinking is that ATP binding and hydrolysis 00:09:40.310 --> 00:10:09.190 results in changes in the hinge geometry 00:10:09.190 --> 00:10:10.950 and that this change in confirmation 00:10:10.950 --> 00:10:14.310 in the hinge with ATP binding and hydrolysis 00:10:14.310 --> 00:10:17.580 allows for conformational change in another subunit here. 00:10:23.390 --> 00:10:26.000 So six rigid bodies connected by six 00:10:26.000 --> 00:10:28.910 hinges, effectively, as opposed to just six 00:10:28.910 --> 00:10:30.650 standalone subunits. 00:10:30.650 --> 00:10:36.140 Each subunit's communicating with one another here. 00:10:36.140 --> 00:10:38.380 So this is pretty complicated, right? 00:10:38.380 --> 00:10:42.500 It's another level of sophistication 00:10:42.500 --> 00:10:45.530 within this hexamer here. 00:10:45.530 --> 00:10:51.380 So what about these loadable and unloadable conformations? 00:10:51.380 --> 00:10:53.000 I've told you that in these crystal 00:10:53.000 --> 00:10:57.680 structures, what's seen often are these four 00:10:57.680 --> 00:11:00.470 loadable and two unloadable subunits 00:11:00.470 --> 00:11:03.240 with a particular arrangement. 00:11:03.240 --> 00:11:06.890 So we can ask the question, do these individual subunits 00:11:06.890 --> 00:11:12.830 maintain the same conformation during these attempts 00:11:12.830 --> 00:11:17.900 to denature and translocate polypeptides? 00:11:17.900 --> 00:11:20.390 So is one subunit just committed to being 00:11:20.390 --> 00:11:25.760 loadable and another subunit committed to being unloadable? 00:11:25.760 --> 00:11:27.800 Or did they switch dynamically? 00:11:27.800 --> 00:11:29.570 And so recently, there were a number 00:11:29.570 --> 00:11:32.390 of studies looking at that. 00:11:32.390 --> 00:11:36.960 And effectively, as of a few years ago, 00:11:36.960 --> 00:11:38.690 many studies suggest-- 00:11:45.371 --> 00:11:59.470 or support switching by a given subunit. 00:12:05.240 --> 00:12:09.770 And they also indicate that every ClpX subunit 00:12:09.770 --> 00:12:14.330 must bind to ATP at some point during these cycles 00:12:14.330 --> 00:12:16.490 for unfolding and translocation. 00:12:25.460 --> 00:12:28.490 But they're not all doing it at the same time. 00:13:00.350 --> 00:13:02.970 So the way to think about this is 00:13:02.970 --> 00:13:05.850 that there's some dynamic interconversion 00:13:05.850 --> 00:13:10.530 between these loadable and unloadable subunits 00:13:10.530 --> 00:13:13.420 within the hexamer, and somehow-- 00:13:13.420 --> 00:13:14.213 yep? 00:13:14.213 --> 00:13:15.630 AUDIENCE: I just want to ask you-- 00:13:15.630 --> 00:13:16.380 ELIZABETH NOLAN: Going to make trouble? 00:13:16.380 --> 00:13:17.210 JOANNE STUBBE: --a question. 00:13:17.210 --> 00:13:18.030 Yeah. 00:13:18.030 --> 00:13:20.550 So when you have all these structures, 00:13:20.550 --> 00:13:22.858 are they all with an ATP analogue? 00:13:22.858 --> 00:13:24.900 ELIZABETH NOLAN: I don't know the answer to that. 00:13:24.900 --> 00:13:25.650 JOANNE STUBBE: OK. 00:13:25.650 --> 00:13:30.000 Because ATP analogues have wide-- 00:13:30.000 --> 00:13:35.730 you guys have already seen ADPNP or ADPCH2P They really 00:13:35.730 --> 00:13:38.940 have very different properties when you study these, the ATPs. 00:13:38.940 --> 00:13:42.290 So if these-- and probably they don't have ATP because they 00:13:42.290 --> 00:13:42.790 probably-- 00:13:42.790 --> 00:13:43.140 ELIZABETH NOLAN: Right. 00:13:43.140 --> 00:13:44.100 They want to get a stable-- 00:13:44.100 --> 00:13:45.690 JOANNE STUBBE: So anyhow, that's something to keep 00:13:45.690 --> 00:13:46.470 in the back of your mind. 00:13:46.470 --> 00:13:48.637 ELIZABETH NOLAN: So just is this an artifact is what 00:13:48.637 --> 00:13:51.750 JoAnne's suggesting from use of a non-hydrolysable ATP 00:13:51.750 --> 00:13:52.490 analogue. 00:13:52.490 --> 00:13:53.820 JOANNE STUBBE: And there's many examples 00:13:53.820 --> 00:13:54.970 of that in the literature. 00:13:54.970 --> 00:13:55.720 Everybody uses it. 00:13:55.720 --> 00:13:58.303 It's just something you need to keep in the back of your mind. 00:13:58.303 --> 00:13:59.460 That's the best we can do. 00:13:59.460 --> 00:14:01.043 ELIZABETH NOLAN: So next week, someone 00:14:01.043 --> 00:14:06.260 should ask during recitation there, for that. 00:14:06.260 --> 00:14:12.810 So what about the mechanical work? 00:14:12.810 --> 00:14:18.300 How this is often depicted, in terms of grabbing and pulling 00:14:18.300 --> 00:14:22.245 on a polypeptide substrate, is via these rigid bodies. 00:14:26.736 --> 00:14:32.010 And we're not going to go into details about this, 00:14:32.010 --> 00:14:37.230 but just to describe the typical cartoon picture effectively, 00:14:37.230 --> 00:14:44.310 imagine we have some polypeptide that needs to enter 00:14:44.310 --> 00:14:45.465 the degradation chamber. 00:14:50.950 --> 00:14:55.300 So those pore loops we heard about that 00:14:55.300 --> 00:15:09.740 are involved in substrate binding 00:15:09.740 --> 00:15:13.670 are in the large domain of ClpX. 00:15:13.670 --> 00:15:15.970 So here we have one large domain, 00:15:15.970 --> 00:15:21.220 and then we can have the small domain of the adjacent subunit 00:15:21.220 --> 00:15:23.360 here. 00:15:23.360 --> 00:15:28.550 And just imagine here we have another large domain 00:15:28.550 --> 00:15:30.710 with its pore loop. 00:15:30.710 --> 00:15:37.370 And then we'd have the adjacent subunit here. 00:15:37.370 --> 00:15:41.900 So effectively, it's thought that these pore loops 00:15:41.900 --> 00:15:46.040 in the large domains grip the substrate and help 00:15:46.040 --> 00:15:49.280 drag the substrate to allow for translocation 00:15:49.280 --> 00:15:51.230 into the degradation chamber. 00:15:51.230 --> 00:15:54.020 So this would be going to the chamber, that direction 00:15:54.020 --> 00:15:55.760 here for that. 00:15:55.760 --> 00:15:58.460 So somehow the ATP binding and hydrolysis 00:15:58.460 --> 00:16:00.410 is allowing this to occur-- 00:16:00.410 --> 00:16:08.060 so to ClpP here for that. 00:16:08.060 --> 00:16:10.940 So next week in recitation, you're 00:16:10.940 --> 00:16:15.890 going to have a real treat because an expert, Reuben, will 00:16:15.890 --> 00:16:18.800 be discussing some single molecule methods that 00:16:18.800 --> 00:16:23.150 have been applied to studying this degradation chamber. 00:16:23.150 --> 00:16:25.970 So bring your questions to him because he really 00:16:25.970 --> 00:16:31.100 knows what is state of the field right now for this. 00:16:31.100 --> 00:16:36.310 So we've talked a lot about how the substrate needs to get in. 00:16:36.310 --> 00:16:37.670 We have the SSRI tag. 00:16:37.670 --> 00:16:40.520 We have all of this ATP consumption unfolding, 00:16:40.520 --> 00:16:42.200 translocation by ClpX. 00:16:42.200 --> 00:16:45.770 And then we talked about the serine protease mechanism 00:16:45.770 --> 00:16:49.130 in terms of how peptides are degraded in the chamber. 00:16:49.130 --> 00:16:53.420 So then the final question just going to touch upon is, 00:16:53.420 --> 00:16:56.810 how does the polypeptide that's been degraded 00:16:56.810 --> 00:16:59.630 get out of the chamber? 00:16:59.630 --> 00:17:09.410 So ClpXP will give products that are 00:17:09.410 --> 00:17:16.010 7 to 8 amino acids in length, so short polypeptides. 00:17:21.790 --> 00:17:23.155 So how are they released? 00:17:27.040 --> 00:17:29.530 And we can think about two possibilities 00:17:29.530 --> 00:17:32.420 for how these polypeptides are released. 00:17:32.420 --> 00:17:38.500 One is that they're released through the axial pores. 00:17:38.500 --> 00:17:42.340 So somehow those pores that allow polypeptide substrate 00:17:42.340 --> 00:17:47.140 to go in also allow product fragments to go out. 00:17:47.140 --> 00:17:52.450 And then the second option is that there's release 00:17:52.450 --> 00:18:06.490 through transient side pores between the ClpP 7-mers. 00:18:09.120 --> 00:18:19.080 So effectively, if we imagine coming back to our ClpP, 00:18:19.080 --> 00:18:21.120 we have a 7-mer-- 00:18:21.120 --> 00:18:30.930 back-to-back 7-mers, do the fragments 00:18:30.930 --> 00:18:36.090 come out, say, of the hole? 00:18:36.090 --> 00:18:39.270 Or somehow do they come out from this region here? 00:18:44.330 --> 00:18:47.460 To the best of my knowledge, this is a bit unclear, 00:18:47.460 --> 00:18:51.210 and I don't think they're mutually exclusive. 00:18:51.210 --> 00:18:52.820 So questions have come up. 00:18:52.820 --> 00:18:55.700 If they're to come out of an axial pore, 00:18:55.700 --> 00:18:58.190 does that mean ClpX has to be dissociated? 00:19:01.100 --> 00:19:03.500 In terms of this equator region, there 00:19:03.500 --> 00:19:06.380 are structures showing that this degradation 00:19:06.380 --> 00:19:07.670 chamber can breathe. 00:19:07.670 --> 00:19:11.900 And there's a picture of that in the posted notes from Friday 00:19:11.900 --> 00:19:14.810 where you can see opening here. 00:19:14.810 --> 00:19:16.580 And there has been some experiments 00:19:16.580 --> 00:19:22.520 done where people have put cysteines in this region 00:19:22.520 --> 00:19:24.150 by site-directed mutagenesis. 00:19:24.150 --> 00:19:26.850 So you can imagine, for instance, 00:19:26.850 --> 00:19:32.210 if you have a cysteine here and a cysteine here, 00:19:32.210 --> 00:19:38.180 and you oxidize to form a disulfide such that those two 00:19:38.180 --> 00:19:40.040 7-mers are locked together. 00:19:40.040 --> 00:19:44.210 You can ask, if we load the chamber with small polypeptides 00:19:44.210 --> 00:19:48.860 and we have these effectively cross-linked by disulfides, 00:19:48.860 --> 00:19:50.900 can the polypeptides get out? 00:19:50.900 --> 00:19:55.270 And then if we reduce this to have them no longer attached 00:19:55.270 --> 00:19:58.660 to one another, do those polypeptides 00:19:58.660 --> 00:20:01.280 stay put or not there? 00:20:01.280 --> 00:20:03.800 Those experiments gave some evidence 00:20:03.800 --> 00:20:08.510 for release of peptides through this region here, 00:20:08.510 --> 00:20:10.760 but there's also evidence for release of peptides 00:20:10.760 --> 00:20:12.680 through the pore. 00:20:12.680 --> 00:20:14.992 And in terms of cartoon depictions. 00:20:14.992 --> 00:20:16.950 In the lecture notes, if you take a close look, 00:20:16.950 --> 00:20:19.620 you'll see that both come out there. 00:20:19.620 --> 00:20:23.360 So I'd say if you're curious about that, 00:20:23.360 --> 00:20:25.430 you can read some of the literature 00:20:25.430 --> 00:20:29.960 and come to some own conclusions. 00:20:29.960 --> 00:20:38.600 One last point on the Clp system before we move on to module 4, 00:20:38.600 --> 00:20:41.600 you should just be aware that there's other Clp family 00:20:41.600 --> 00:20:43.340 members. 00:20:43.340 --> 00:20:50.990 So not only ClpX and P. And so in the Clp system-- 00:20:50.990 --> 00:20:53.240 actually, I'm going to make one other point after this 00:20:53.240 --> 00:20:56.165 too, about degradation chambers. 00:21:01.710 --> 00:21:07.950 So there are players ClpA, ClpB, in addition to ClpX. 00:21:10.860 --> 00:21:20.945 So these are all three different AAA+ ATPases. 00:21:24.600 --> 00:21:28.770 And you've actually encountered ClpB last week. 00:21:28.770 --> 00:21:33.450 So this is HSP 100, which came up in question 2 on the exam 00:21:33.450 --> 00:21:36.210 there by another name. 00:21:36.210 --> 00:21:39.380 And then in addition to ClpP, there's 00:21:39.380 --> 00:21:46.230 also ClpS and some other players here for that. 00:21:46.230 --> 00:21:52.200 They each have their own personality within protein 00:21:52.200 --> 00:21:55.430 quality control here for that. 00:22:02.540 --> 00:22:06.890 And then we've only looked at this degradation chamber 00:22:06.890 --> 00:22:09.642 from bacteria. 00:22:09.642 --> 00:22:11.100 You might want to ask the question, 00:22:11.100 --> 00:22:16.380 what happens in other organisms? 00:22:16.380 --> 00:22:20.420 And the answer is that the complexity varies and systems 00:22:20.420 --> 00:22:25.280 become tremendously more complex as you move from bacteria 00:22:25.280 --> 00:22:27.360 into eukaryotes there. 00:22:27.360 --> 00:22:32.670 And so if we consider the different degradation chambers, 00:22:32.670 --> 00:22:34.370 what do we see? 00:22:34.370 --> 00:22:38.930 So we find these proteasomes in all forms of life. 00:22:38.930 --> 00:22:42.020 And as I just said, the level of complexity 00:22:42.020 --> 00:22:44.820 varies depending on the organism. 00:22:44.820 --> 00:22:51.410 And so what we've seen with ClpP is the most simple system 00:22:51.410 --> 00:22:58.380 where we have two rings that have only one type of subunit. 00:22:58.380 --> 00:23:00.570 So just say E. coli. 00:23:00.570 --> 00:23:01.730 One type of subunit. 00:23:17.300 --> 00:23:20.000 What happens if we go to archae? 00:23:20.000 --> 00:23:23.570 We find that we have four rings, each of which is 7-mer. 00:23:34.770 --> 00:23:39.160 And these four rings include two different types of subunits. 00:23:39.160 --> 00:23:41.430 So I'll call these alpha and beta. 00:23:41.430 --> 00:23:46.740 So what we find is that there's a 7-mer of 7 alpha subunits, 00:23:46.740 --> 00:23:50.610 then 7-mers that have 7 beta subunits, and here 00:23:50.610 --> 00:23:51.555 a 7-mer with alpha. 00:23:55.800 --> 00:24:04.465 So we see two types of subunit and four rings. 00:24:11.080 --> 00:24:14.080 So then what about yeast? 00:24:14.080 --> 00:24:15.460 Tremendously complex. 00:24:15.460 --> 00:24:20.440 So we have this architecture again 00:24:20.440 --> 00:24:28.030 of four rings, an organized alpha, beta, beta, alpha. 00:24:33.720 --> 00:24:42.300 But what we find in this case is that in each of these-- 00:24:42.300 --> 00:24:44.580 I'm not going to draw it like that, but each of these 00:24:44.580 --> 00:24:45.830 have seven different subunits. 00:24:55.250 --> 00:24:56.960 There's a depiction of this in the notes. 00:25:08.770 --> 00:25:14.550 So just imagine-- how does this get assembled? 00:25:14.550 --> 00:25:15.810 I have no clue. 00:25:15.810 --> 00:25:18.300 But somehow each of these heptamers 00:25:18.300 --> 00:25:21.000 has to be assembled with seven different subunits. 00:25:21.000 --> 00:25:23.910 And then they're put together in this series of four rings. 00:25:23.910 --> 00:25:26.370 And then as you'll see after spring break in JoAnne's 00:25:26.370 --> 00:25:29.340 section, the eukaryotic proteasome 00:25:29.340 --> 00:25:33.600 has this 19S regulatory particle that's 00:25:33.600 --> 00:25:36.840 involved in recognizing condemned proteins that 00:25:36.840 --> 00:25:38.760 have polyubiquitin chains. 00:25:38.760 --> 00:25:44.020 And compared to the ClpX ATPase, it's much, much more complex. 00:25:44.020 --> 00:25:45.630 So there's many different proteins 00:25:45.630 --> 00:25:50.220 that constitute this necessary part of the machine. 00:25:50.220 --> 00:25:54.450 But there is a hexamer, ATPase hexamer, within there 00:25:54.450 --> 00:25:57.660 to facilitate translocation of the polypeptide 00:25:57.660 --> 00:26:01.260 into the degradation chamber. 00:26:01.260 --> 00:26:05.222 So some of this will come back again 00:26:05.222 --> 00:26:06.555 in the latter half of the class. 00:26:09.170 --> 00:26:14.980 So with that, we're going to close on degradation and move 00:26:14.980 --> 00:26:21.400 into module 4, which is focused on macromolecular machines that 00:26:21.400 --> 00:26:25.120 are involved in the biosynthesis of natural products, 00:26:25.120 --> 00:26:29.360 specifically polyketides and nonribosomal peptides. 00:26:29.360 --> 00:26:32.920 And so we're completely taking a loop back 00:26:32.920 --> 00:26:35.920 to thinking about a biological polymerization, 00:26:35.920 --> 00:26:38.470 like what we were thinking with the ribosome 00:26:38.470 --> 00:26:42.700 from the process of breaking down a polypeptide. 00:26:42.700 --> 00:26:45.760 And so where are we going? 00:26:49.510 --> 00:26:51.220 We can think about assembly lines, 00:26:51.220 --> 00:26:54.160 although this is a helpful way on the board 00:26:54.160 --> 00:26:55.413 to think about these systems. 00:26:55.413 --> 00:26:57.080 But it's not really what they look like. 00:26:57.080 --> 00:27:00.520 And you'll learn about that in recitation this week. 00:27:00.520 --> 00:27:01.020 Yeah? 00:27:01.020 --> 00:27:02.844 AUDIENCE: Could you explain the interaction 00:27:02.844 --> 00:27:06.057 between ATP and the hinge area? 00:27:06.057 --> 00:27:06.890 ELIZABETH NOLAN: OK. 00:27:06.890 --> 00:27:11.510 So the ATP binding site is just rewinding here 00:27:11.510 --> 00:27:13.130 in that hinge region. 00:27:13.130 --> 00:27:15.500 And there's going to be conformational change 00:27:15.500 --> 00:27:19.100 in the hinge with ATP binding and hydrolysis there. 00:27:19.100 --> 00:27:23.420 And that's sufficient in terms of the level of detail 00:27:23.420 --> 00:27:23.960 for this. 00:27:23.960 --> 00:27:28.340 But the main thing to keep in mind, each subunit binds ATP. 00:27:28.340 --> 00:27:31.310 But on the basis of the information gathered 00:27:31.310 --> 00:27:33.620 with the caveats JoAnne brought up, 00:27:33.620 --> 00:27:38.600 different subunits bind ATP at different times in the cycle. 00:27:38.600 --> 00:27:39.512 AUDIENCE: OK. 00:27:39.512 --> 00:27:41.180 Thank you. 00:27:41.180 --> 00:27:43.490 ELIZABETH NOLAN: And changes in this subunit, 00:27:43.490 --> 00:27:45.530 conformational changes that result from that, 00:27:45.530 --> 00:27:49.945 can be translated to the next door subunit here. 00:27:49.945 --> 00:27:51.310 AUDIENCE: OK. 00:27:51.310 --> 00:27:53.190 ELIZABETH NOLAN: OK. 00:27:53.190 --> 00:27:56.340 So where are we going? 00:27:56.340 --> 00:28:02.100 By a week from now, you should have a good handle 00:28:02.100 --> 00:28:04.980 on how to think about the biosynthesis of structures 00:28:04.980 --> 00:28:08.310 like erythromycin, of penicillin. 00:28:08.310 --> 00:28:12.450 These are products of assembly lines. 00:28:12.450 --> 00:28:16.950 And so where we'll go is with a brief overview of fatty acid 00:28:16.950 --> 00:28:20.880 biosynthesis and then look into polyketide synthase 00:28:20.880 --> 00:28:25.510 and nonribosomal peptide synthetase assembly lines here. 00:28:25.510 --> 00:28:26.970 And then some case studies. 00:28:26.970 --> 00:28:32.850 So on the topic of ATP, where we just went back to with ClpX, 00:28:32.850 --> 00:28:36.090 just taking a look here, what do you 00:28:36.090 --> 00:28:40.450 know about these systems in ATP by the names? 00:28:40.450 --> 00:28:49.400 This is just a little language use and definition. 00:28:49.400 --> 00:28:52.832 So there's a subtle difference here. 00:28:52.832 --> 00:28:53.998 What's the difference? 00:28:53.998 --> 00:28:55.540 AUDIENCE: Synthase versus synthetase? 00:28:55.540 --> 00:28:56.300 ELIZABETH NOLAN: Yeah. 00:28:56.300 --> 00:28:58.217 And what does that tell you right off the bat? 00:29:01.112 --> 00:29:02.086 About ATP. 00:29:14.750 --> 00:29:18.380 So it's a subtlety, right? 00:29:18.380 --> 00:29:20.510 Synthase is a general term. 00:29:20.510 --> 00:29:23.380 Synthetase indicates ATP is involved. 00:29:23.380 --> 00:29:25.485 So as we'll see, these nonribosomal peptide 00:29:25.485 --> 00:29:27.662 synthetases employ ATP. 00:29:27.662 --> 00:29:29.870 And we're going to see chemistry very similar to what 00:29:29.870 --> 00:29:32.840 you saw with the aminoacyl-tRNA synthetases 00:29:32.840 --> 00:29:35.720 in terms of activating amino acid monomers. 00:29:35.720 --> 00:29:40.640 But in this case, the machine is forming a nonribosomal peptide 00:29:40.640 --> 00:29:45.320 rather than a ribosomal peptide here. 00:29:45.320 --> 00:29:49.790 If you are not familiar with fatty acid biosynthesis, 00:29:49.790 --> 00:29:52.550 I highly encourage you to go do some review, 00:29:52.550 --> 00:29:55.880 either from your 5.07 notes last term if you were in the class 00:29:55.880 --> 00:29:57.740 or from a biochemistry book. 00:29:57.740 --> 00:30:00.860 And there'll be some additional slides of overview information 00:30:00.860 --> 00:30:01.820 posted online. 00:30:01.820 --> 00:30:04.400 So we'll just touch upon it today but not 00:30:04.400 --> 00:30:08.090 go into tremendous detail here. 00:30:08.090 --> 00:30:12.155 So what are our questions for this module? 00:30:12.155 --> 00:30:14.480 I think for most everyone in the room, 00:30:14.480 --> 00:30:18.050 this module will contain the most new information 00:30:18.050 --> 00:30:19.550 from the standpoint of a new system 00:30:19.550 --> 00:30:22.740 compared to what we've talked about so far. 00:30:22.740 --> 00:30:27.500 So what are polyketides and how are these molecules 00:30:27.500 --> 00:30:31.430 biosynthesized by polyketide synthases? 00:30:31.430 --> 00:30:34.130 What are nonribosomal peptides and how are they 00:30:34.130 --> 00:30:37.640 made by these machines called nonribosomal peptide 00:30:37.640 --> 00:30:38.930 synthetases? 00:30:38.930 --> 00:30:42.230 And what we're going to look at is the assembly line 00:30:42.230 --> 00:30:47.420 organization, so effectively the organization of domains that 00:30:47.420 --> 00:30:52.620 provide these linear polymers. 00:30:52.620 --> 00:30:57.050 So what is the assembly line organization and logic for PKS? 00:30:57.050 --> 00:30:59.720 And likewise for NRPS. 00:30:59.720 --> 00:31:02.540 And then we can ask, how can a given assembly 00:31:02.540 --> 00:31:07.250 line for a given PKS or NRPS natural product 00:31:07.250 --> 00:31:11.390 be basically predicted from the structure 00:31:11.390 --> 00:31:12.530 of the natural product? 00:31:12.530 --> 00:31:14.900 So you should be able to work back and forth in terms 00:31:14.900 --> 00:31:16.550 of looking at a structure and coming up 00:31:16.550 --> 00:31:19.670 with a biosynthetic prediction and also seeing 00:31:19.670 --> 00:31:22.190 biosynthetic machinery and getting a sense as to what 00:31:22.190 --> 00:31:27.290 that small molecule metabolite's backbone might look like. 00:31:27.290 --> 00:31:30.290 How are these studied experimentally? 00:31:30.290 --> 00:31:32.660 And we'll look at the biosynthesis 00:31:32.660 --> 00:31:35.870 of a molecule called enterobactin as a case study. 00:31:35.870 --> 00:31:38.240 And so one thing I'll just point out right now 00:31:38.240 --> 00:31:42.380 is that these synthases and synthetases do not 00:31:42.380 --> 00:31:43.880 look like an assembly line. 00:31:43.880 --> 00:31:46.910 And we'll draw domains in a linear order which really 00:31:46.910 --> 00:31:50.000 facilitates thinking about the chemistry, 00:31:50.000 --> 00:31:54.710 but the structures are not just a line of domains or proteins 00:31:54.710 --> 00:31:56.430 next door to one another. 00:31:56.430 --> 00:31:58.370 And this week in recitation, you'll 00:31:58.370 --> 00:32:04.370 get to see some cryo-EM studies on fatty acid synthase and 00:32:04.370 --> 00:32:06.650 related machines there which will give you 00:32:06.650 --> 00:32:11.310 a sense of their dynamics. 00:32:11.310 --> 00:32:14.120 So just a review. 00:32:14.120 --> 00:32:18.260 If we think about template-dependent 00:32:18.260 --> 00:32:20.720 polymerizations in biology, we're 00:32:20.720 --> 00:32:24.890 all familiar with DNA replication, transcription, 00:32:24.890 --> 00:32:27.050 and translation. 00:32:27.050 --> 00:32:29.690 And what you'll see in this unit is 00:32:29.690 --> 00:32:32.600 that these template-driven polymerizations 00:32:32.600 --> 00:32:37.730 occur in the biosynthesis of natural products here. 00:32:37.730 --> 00:32:39.740 And effectively, these assembly lines, in a way, 00:32:39.740 --> 00:32:42.080 provide this template. 00:32:42.080 --> 00:32:45.260 So they're small molecules being biosynthesized 00:32:45.260 --> 00:32:51.840 by microbes using some pretty amazing machinery. 00:32:51.840 --> 00:32:53.780 So when we think about template-driven 00:32:53.780 --> 00:32:57.950 polymerizations, we think about an initiation process, 00:32:57.950 --> 00:33:00.560 elongation process, and termination. 00:33:00.560 --> 00:33:03.680 We saw that with a translation cycle. 00:33:03.680 --> 00:33:10.160 And we'll see the same type of systems here. 00:33:10.160 --> 00:33:14.030 So what does some of these structures look like? 00:33:14.030 --> 00:33:16.490 Here are just some examples on the top 00:33:16.490 --> 00:33:21.440 of polyketides, two examples. 00:33:21.440 --> 00:33:24.950 They look very different at first glance, and they are. 00:33:24.950 --> 00:33:27.350 So we have tetracycline. 00:33:27.350 --> 00:33:30.830 We have four fused 6-membered rings. 00:33:30.830 --> 00:33:34.610 It's an aromatic polyketide, an antibiotic. 00:33:34.610 --> 00:33:39.410 We have this erythromycin here, which is a macrolide. 00:33:39.410 --> 00:33:42.350 We encountered macrolines in the translation section 00:33:42.350 --> 00:33:46.850 because they bind the ribosome, another type of antibiotic. 00:33:46.850 --> 00:33:51.890 If we look at some nonribosomal peptides, all of that 00:33:51.890 --> 00:33:53.330 can be used clinically. 00:33:53.330 --> 00:33:54.980 We see the penicillins. 00:33:54.980 --> 00:34:01.940 So we have a 4 or 5 fused ring system here, a beta-lactam. 00:34:01.940 --> 00:34:06.320 This comes from three amino acid building blocks initially. 00:34:06.320 --> 00:34:08.060 We have vancomycin. 00:34:08.060 --> 00:34:10.730 This is an antibiotic of last resort. 00:34:10.730 --> 00:34:14.350 And this structure looks really quite complicated, 00:34:14.350 --> 00:34:19.540 but what we'll see is that it's based on seven 00:34:19.540 --> 00:34:21.050 proteogenic amino acids. 00:34:21.050 --> 00:34:23.650 So it's the 7-mer peptide backbone 00:34:23.650 --> 00:34:29.530 that gives rise to this structure here for that. 00:34:29.530 --> 00:34:31.270 And then we see there's some sugars, 00:34:31.270 --> 00:34:35.920 so these can be put on by other enzymes here. 00:34:35.920 --> 00:34:40.090 So on top we see a lot of ketones and OH groups. 00:34:40.090 --> 00:34:44.710 Those are good hints that maybe polyketide logic is being used. 00:34:44.710 --> 00:34:47.510 Here we see a number of peptide bonds, 00:34:47.510 --> 00:34:53.550 amide bonds, a good indicator of NRPS at play. 00:34:53.550 --> 00:34:58.860 And here, just to point out, these systems get very complex. 00:34:58.860 --> 00:35:02.250 And there's natural products out there 00:35:02.250 --> 00:35:05.430 that are biosynthesized from a combination 00:35:05.430 --> 00:35:10.260 of polyketide synthase logic and nonribosomal peptide synthetase 00:35:10.260 --> 00:35:11.820 logic here. 00:35:11.820 --> 00:35:15.960 These include molecules like yersiniabactin. 00:35:15.960 --> 00:35:19.080 This is an iron chelator produced by Yersinia pestis, 00:35:19.080 --> 00:35:22.640 and some pathogenic E. coli, this immunosuppressant 00:35:22.640 --> 00:35:26.070 rapamycin as examples. 00:35:26.070 --> 00:35:28.860 So as we move forward, I put a lot 00:35:28.860 --> 00:35:32.520 of structures of small molecule metabolites in the slides. 00:35:32.520 --> 00:35:34.170 You can go back and use them as a way 00:35:34.170 --> 00:35:35.910 to study and try to make predictions 00:35:35.910 --> 00:35:39.120 about what is the machinery at play, 00:35:39.120 --> 00:35:42.120 for instance, to give all of these heterocycles? 00:35:42.120 --> 00:35:43.860 How are those made? 00:35:43.860 --> 00:35:47.310 We'll see the assembly line does that. 00:35:47.310 --> 00:35:51.060 So what organisms produce these molecules? 00:35:51.060 --> 00:35:54.870 Largely, bacteria and fungi. 00:35:54.870 --> 00:35:59.550 And there are some correlations out there, I'll just point out, 00:35:59.550 --> 00:36:05.480 related to genome size and the number 00:36:05.480 --> 00:36:07.970 of metabolites being made. 00:36:07.970 --> 00:36:12.380 So bioinformatics guides a lot of current studies 00:36:12.380 --> 00:36:15.320 of the biosynthesis of these types of molecules. 00:36:15.320 --> 00:36:18.500 So you can imagine that you sequence a genome. 00:36:18.500 --> 00:36:22.100 You have some information about gene clusters. 00:36:22.100 --> 00:36:23.960 So these are groups of genes where 00:36:23.960 --> 00:36:28.100 the proteins work together to biosynthesize the molecule. 00:36:28.100 --> 00:36:32.330 And often, the genes that encode proteins in these metabolites 00:36:32.330 --> 00:36:34.670 are clustered. 00:36:34.670 --> 00:36:38.750 And so bioinformatics approaches can help find these. 00:36:38.750 --> 00:36:44.060 What's found is that for bacteria, some phyla 00:36:44.060 --> 00:36:48.950 are more prolific producers of these molecules than others. 00:36:48.950 --> 00:36:53.090 And what's been shown in a general way 00:36:53.090 --> 00:36:56.480 is that organisms with small genomes-- 00:36:56.480 --> 00:36:58.700 so something like E. coli-- 00:36:58.700 --> 00:37:01.730 produce fewer of these metabolites. 00:37:01.730 --> 00:37:03.110 That's not to say none. 00:37:03.110 --> 00:37:05.630 So enterobactin, which we'll look at for a case study, 00:37:05.630 --> 00:37:07.640 is made by E. coli. 00:37:07.640 --> 00:37:09.920 But they don't make as many. 00:37:09.920 --> 00:37:17.130 And effectively, organisms with larger genomes produce more. 00:37:17.130 --> 00:37:18.860 And so here is just a correlation 00:37:18.860 --> 00:37:20.750 between the number of genes. 00:37:20.750 --> 00:37:22.640 And the genome size of the organism 00:37:22.640 --> 00:37:28.940 where they see around 3 Mb, there's a switch here. 00:37:28.940 --> 00:37:30.445 Often, these molecules-- yeah? 00:37:30.445 --> 00:37:31.820 AUDIENCE: Is there any hypotheses 00:37:31.820 --> 00:37:34.070 about an evolutionary driving factor 00:37:34.070 --> 00:37:35.630 for the development of this machinery 00:37:35.630 --> 00:37:39.328 and why it correlates to genome size? 00:37:39.328 --> 00:37:41.120 ELIZABETH NOLAN: If there is, I don't know. 00:37:41.120 --> 00:37:45.050 I don't think about evolution very well, quite frankly. 00:37:45.050 --> 00:37:48.980 What is thought is that many of these molecules 00:37:48.980 --> 00:37:51.290 are thought to be involved in defense 00:37:51.290 --> 00:37:54.290 and that an organism with a smaller genome size 00:37:54.290 --> 00:37:55.730 uses other strategies. 00:37:55.730 --> 00:37:59.150 And so for instance, E. coli, which 00:37:59.150 --> 00:38:01.460 I cited as a small genome, will use 00:38:01.460 --> 00:38:05.670 a number of ribosomal peptides as defense molecules 00:38:05.670 --> 00:38:08.510 that get post-translationally modified after the fact. 00:38:08.510 --> 00:38:12.170 But why that organism chooses to do that versus say something 00:38:12.170 --> 00:38:16.430 like Streptomyces that produces many, many different natural 00:38:16.430 --> 00:38:19.190 products, I'm not sure about that. 00:38:24.170 --> 00:38:29.600 So let's look at an example of a gene cluster, 00:38:29.600 --> 00:38:33.590 just so you get a sense of how much machinery 00:38:33.590 --> 00:38:39.020 is required to do the full biosynthesis of a molecule. 00:38:39.020 --> 00:38:43.190 So this is for a nonribosomal peptide shown here. 00:38:43.190 --> 00:38:45.080 It has some structural similarities 00:38:45.080 --> 00:38:49.520 to the vancomycin we saw on a prior slide, 00:38:49.520 --> 00:38:52.820 and it is a member of the vancomycin family. 00:38:52.820 --> 00:38:57.530 So this gene cluster for the biosynthesis of this metabolite 00:38:57.530 --> 00:39:02.330 contains 30 different genes and is depicted here. 00:39:02.330 --> 00:39:05.210 So each one of these arrows indicates an open reading 00:39:05.210 --> 00:39:06.330 frame. 00:39:06.330 --> 00:39:10.860 So each one begins with a start, ends with a stop codon. 00:39:10.860 --> 00:39:14.300 And it's assumed to be the coding sequence of the gene. 00:39:14.300 --> 00:39:17.870 And so what is encoded in these 30 genes? 00:39:17.870 --> 00:39:21.290 Well, first there are the genes for what 00:39:21.290 --> 00:39:23.270 we call the assembly line. 00:39:23.270 --> 00:39:25.610 And if it isn't clear what assembly line means, 00:39:25.610 --> 00:39:28.430 as we move forward through this week, it will be. 00:39:28.430 --> 00:39:33.650 So there's genes required to make the 7-mer polypeptide 00:39:33.650 --> 00:39:35.060 backbone. 00:39:35.060 --> 00:39:38.750 There's genes required for modification of the backbone. 00:39:38.750 --> 00:39:43.020 So how do these sugars get attached, for instance? 00:39:43.020 --> 00:39:45.650 Those are going to be some tailoring enzymes. 00:39:45.650 --> 00:39:47.640 And then if you take a close look, 00:39:47.640 --> 00:39:51.500 there's a number of non-proteinogenic amino acids 00:39:51.500 --> 00:39:53.720 in this molecule, and that means they 00:39:53.720 --> 00:39:55.380 have to come from somewhere. 00:39:55.380 --> 00:39:57.890 And so this gene cluster also includes 00:39:57.890 --> 00:40:02.810 genes that are required for the biosynthesis of those monomers. 00:40:02.810 --> 00:40:05.600 So there's a lot of effort going in to making 00:40:05.600 --> 00:40:09.140 this molecule by some organism. 00:40:09.140 --> 00:40:11.930 And so presumably, under some set of conditions, 00:40:11.930 --> 00:40:12.800 it's important. 00:40:16.390 --> 00:40:24.130 So moving towards the chemistry, with that background in hand, 00:40:24.130 --> 00:40:27.490 what are some points to make? 00:40:27.490 --> 00:40:34.660 So what we'll learn and see is that the assembly lines that 00:40:34.660 --> 00:40:39.250 produce the polyketides and nonribosomal peptides 00:40:39.250 --> 00:40:41.240 are macromolecular machines. 00:40:41.240 --> 00:40:44.440 So there's dedicated macromolecular machines 00:40:44.440 --> 00:40:49.960 for the biosynthesis of these secondary metabolites. 00:40:49.960 --> 00:40:52.510 And so what are secondary metabolites 00:40:52.510 --> 00:40:56.120 versus a primary metabolite? 00:40:56.120 --> 00:40:57.970 So what's a primary metabolite? 00:41:08.530 --> 00:41:11.930 AUDIENCE: I'm not even totally sure how to define metabolites. 00:41:11.930 --> 00:41:14.470 Isn't metabolites what goes in? 00:41:14.470 --> 00:41:17.180 Or what comes out? 00:41:20.269 --> 00:41:21.311 ELIZABETH NOLAN: Rebecca? 00:41:21.311 --> 00:41:24.490 AUDIENCE: Or easily produced directly from the materials 00:41:24.490 --> 00:41:27.400 the cell's consuming? 00:41:27.400 --> 00:41:30.820 ELIZABETH NOLAN: So presumably, the cell 00:41:30.820 --> 00:41:33.160 needs to get materials to biosynthesize 00:41:33.160 --> 00:41:35.320 the secondary metabolites too, right? 00:41:35.320 --> 00:41:37.300 Somewhere, these amino acid monomers 00:41:37.300 --> 00:41:40.660 or the monomers that are used for polyketide synthetase 00:41:40.660 --> 00:41:41.370 need to-- 00:41:41.370 --> 00:41:43.690 they'd have to come from somewhere, right? 00:41:43.690 --> 00:41:46.550 So are primary metabolites important for growth? 00:41:46.550 --> 00:41:47.225 AUDIENCE: Yes. 00:41:47.225 --> 00:41:48.100 ELIZABETH NOLAN: Yes. 00:41:48.100 --> 00:41:50.530 Development? 00:41:50.530 --> 00:41:51.270 Reproduction? 00:41:51.270 --> 00:41:52.170 AUDIENCE: Yes. 00:41:52.170 --> 00:41:53.378 ELIZABETH NOLAN: Yeah, right. 00:41:53.378 --> 00:41:54.960 Under normal conditions, right? 00:41:54.960 --> 00:41:57.540 We're in trouble if we don't have our primary metabolites 00:41:57.540 --> 00:42:02.070 there, whether they're ingested or biosynthesized. 00:42:02.070 --> 00:42:03.960 What about a secondary metabolite? 00:42:03.960 --> 00:42:05.100 Just taking that-- 00:42:05.100 --> 00:42:06.195 AUDIENCE: I'm guessing it's not necessary. 00:42:06.195 --> 00:42:07.854 AUDIENCE: --something we can make from primary metabolites? 00:42:07.854 --> 00:42:09.140 ELIZABETH NOLAN: No. 00:42:09.140 --> 00:42:10.650 Well, you can. 00:42:10.650 --> 00:42:11.770 You can. 00:42:11.770 --> 00:42:13.393 So a secondary-- 00:42:13.393 --> 00:42:14.310 AUDIENCE: --necessary? 00:42:14.310 --> 00:42:15.227 ELIZABETH NOLAN: Yeah. 00:42:15.227 --> 00:42:20.100 A secondary metabolite is not required for normal growth, 00:42:20.100 --> 00:42:22.290 development, reproduction. 00:42:22.290 --> 00:42:27.690 So for some reason, under some circumstances of need, 00:42:27.690 --> 00:42:30.250 these secondary metabolites get produced. 00:42:30.250 --> 00:42:33.840 So for some of these antibiotic molecules, maybe 00:42:33.840 --> 00:42:36.480 the organism needs to defend itself. 00:42:36.480 --> 00:42:39.450 In the case of enterobactin or yersiniabactin, 00:42:39.450 --> 00:42:41.880 maybe that organism needs iron. 00:42:41.880 --> 00:42:43.770 And so it's producing a molecule that 00:42:43.770 --> 00:42:47.880 will help it obtain that there. 00:42:47.880 --> 00:42:51.310 So what is going on? 00:42:51.310 --> 00:42:54.090 We've seen some pretty complex molecules. 00:42:54.090 --> 00:42:59.100 What we're going to see is that these assembly lines convert 00:42:59.100 --> 00:43:03.360 simple acid monomers, if it's a polyketide synthase 00:43:03.360 --> 00:43:06.700 or amino acid monomers for a nonribosomal peptide 00:43:06.700 --> 00:43:09.810 synthetase, into linear polymers. 00:43:09.810 --> 00:43:13.350 So we're going to look at template-driven polymerizations 00:43:13.350 --> 00:43:16.260 that initially give linear polymers. 00:43:16.260 --> 00:43:19.440 And in the case of PKS, this is very similar 00:43:19.440 --> 00:43:22.200 to fatty acid biosynthesis. 00:43:22.200 --> 00:43:25.530 What we see is that the assembly lines 00:43:25.530 --> 00:43:29.100 allow for iterative additions of malonyl and methylmalonyl 00:43:29.100 --> 00:43:30.060 units. 00:43:30.060 --> 00:43:34.200 And they catalyze carbon-carbon bond formations. 00:43:34.200 --> 00:43:38.730 In the case of nonribosomal peptide synthetases, 00:43:38.730 --> 00:43:42.990 what we'll see is that these allow for condensations 00:43:42.990 --> 00:43:45.810 of amino acids to form peptide bonds 00:43:45.810 --> 00:43:49.510 and effectively form nonribosomal polypeptides. 00:43:49.510 --> 00:43:53.070 So polypeptide synthesis without the ribosome. 00:43:53.070 --> 00:43:57.480 So even though the PKS and NRPS are forming a different type 00:43:57.480 --> 00:44:01.770 of bond and that requires different chemistry, what 00:44:01.770 --> 00:44:07.050 we'll see is that they use very similar logic. 00:44:07.050 --> 00:44:11.730 And just getting the logic sorted out initially 00:44:11.730 --> 00:44:13.710 makes life much easier down the road. 00:44:13.710 --> 00:44:17.940 So take some time to look over the depictions in the notes 00:44:17.940 --> 00:44:21.100 outside of class as we go forward. 00:44:21.100 --> 00:44:25.620 So these assembly lines use acyl or aminoacyl thioesters 00:44:25.620 --> 00:44:29.040 as the activated monomer units. 00:44:29.040 --> 00:44:32.310 So then how do we get from this linear polypeptide 00:44:32.310 --> 00:44:34.140 to some more complex structure? 00:44:36.750 --> 00:44:40.890 The short message on that is that the, quote, "polymers" 00:44:40.890 --> 00:44:42.650 that are produced-- 00:44:42.650 --> 00:44:44.040 and they may be short, right? 00:44:44.040 --> 00:44:47.790 We just saw-- they are short, 7 amino acids for vancomycin. 00:44:47.790 --> 00:44:50.460 They can undergo further elaboration 00:44:50.460 --> 00:44:52.620 to give these complex structures. 00:44:52.620 --> 00:44:56.490 So there can be tailoring enzymes 00:44:56.490 --> 00:45:01.200 that work on the products of the assembly line. 00:45:01.200 --> 00:45:03.930 Or there can be domains in the assembly line that 00:45:03.930 --> 00:45:08.130 give additional activities that allow for methylation 00:45:08.130 --> 00:45:11.940 or cyclization here. 00:45:11.940 --> 00:45:19.150 So we can think about fatty acid synthase as a paradigm here. 00:45:19.150 --> 00:45:22.560 And so if we think about fatty acid 00:45:22.560 --> 00:45:30.330 biosynthesis making some molecule like this oil here, 00:45:30.330 --> 00:45:34.830 just as brief overview in the last few minutes of class. 00:45:34.830 --> 00:45:38.300 Fatty acids are synthesized by FAS. 00:45:38.300 --> 00:45:41.580 And what happens is that there's elongation 00:45:41.580 --> 00:45:45.060 by one unit at a time. 00:45:45.060 --> 00:45:47.620 And each unit provides two carbons. 00:45:47.620 --> 00:45:51.040 So there's two carbon atoms per elongation. 00:45:51.040 --> 00:45:53.760 And so hopefully you're all familiar with two ways 00:45:53.760 --> 00:45:56.430 to form a carbon-carbon bond, at least related 00:45:56.430 --> 00:46:00.840 to biochemistry, one of which is Claisen condensations. 00:46:00.840 --> 00:46:04.410 So Claisen condensations allow for carbon-carbon bond 00:46:04.410 --> 00:46:09.420 formation and join the units. 00:46:09.420 --> 00:46:12.355 To keep in mind, the monomers are always 00:46:12.355 --> 00:46:16.350 thioesters, not oxoesters. 00:46:16.350 --> 00:46:20.550 And for fatty acid biosynthesis, the two monomer units 00:46:20.550 --> 00:46:22.090 are shown here. 00:46:22.090 --> 00:46:26.880 So we have a starter and an extender, acetyl CoA or malonyl 00:46:26.880 --> 00:46:29.070 CoA here. 00:46:29.070 --> 00:46:31.620 And here we have coenzyme A. 00:46:31.620 --> 00:46:35.665 So just as a brief review, if we think about these monomer 00:46:35.665 --> 00:46:36.165 units-- 00:47:01.730 --> 00:47:07.862 so here we have acetyl CoA. 00:47:14.620 --> 00:47:28.680 So what can we say about this guy here, in this thioester? 00:47:45.950 --> 00:47:50.280 So is this acidic or not? 00:47:57.400 --> 00:47:59.090 Compared to an oxoester. 00:48:04.000 --> 00:48:06.890 How many of you have heard about fatty acid biosynthesis? 00:48:10.744 --> 00:48:13.700 AUDIENCE: [INAUDIBLE] 00:48:13.700 --> 00:48:17.230 ELIZABETH NOLAN: So why are thioesters used and not 00:48:17.230 --> 00:48:17.769 oxoesters? 00:48:17.769 --> 00:48:19.436 AUDIENCE: [INAUDIBLE] use the other end? 00:48:25.497 --> 00:48:26.330 ELIZABETH NOLAN: OK. 00:48:26.330 --> 00:48:30.260 So we'll go into a little more detail on Friday 00:48:30.260 --> 00:48:32.840 to make sure the chemistry is straight here 00:48:32.840 --> 00:48:34.700 because I'm not certain it is. 00:48:34.700 --> 00:48:36.188 So-- 00:48:36.188 --> 00:48:42.447 AUDIENCE: Is oxoester referring to not that [INAUDIBLE]---- 00:48:42.447 --> 00:48:43.280 ELIZABETH NOLAN: OK. 00:48:43.280 --> 00:49:02.710 So for Friday, think about a thioester versus an oxoester, 00:49:02.710 --> 00:49:05.080 and how do properties differ? 00:49:05.080 --> 00:49:09.970 And why might we want to be using thioesters? 00:49:09.970 --> 00:49:12.340 And also review the Claisen condensation 00:49:12.340 --> 00:49:14.320 because that's the chemistry that's 00:49:14.320 --> 00:49:17.230 going to be happening to form the carbon-carbon bonds 00:49:17.230 --> 00:49:22.270 in the fatty acid synthase and in the polyketide synthases. 00:49:22.270 --> 00:49:25.510 And what we're going to see is that the monomers in each case, 00:49:25.510 --> 00:49:26.860 they're tethered as thioesters. 00:49:26.860 --> 00:49:28.450 So why is that? 00:49:33.750 --> 00:49:38.450 And I will turn around and point at somebody, 00:49:38.450 --> 00:49:40.320 and you can let us know. 00:49:40.320 --> 00:49:42.540 Are you excited? 00:49:42.540 --> 00:49:43.110 OK. 00:49:43.110 --> 00:49:46.140 So you're off the hook for Wednesday. 00:49:46.140 --> 00:49:49.970 I need to be out of town, and I'll see you on Friday.