WEBVTT 00:00:00.500 --> 00:00:02.830 The following content is provided under a Creative 00:00:02.830 --> 00:00:04.370 Commons license. 00:00:04.370 --> 00:00:06.670 Your support will help MIT OpenCourseWare 00:00:06.670 --> 00:00:11.030 continue to offer high-quality educational resources for free. 00:00:11.030 --> 00:00:13.660 To make a donation or view additional materials 00:00:13.660 --> 00:00:17.610 from hundreds of MIT courses, visit MIT OpenCourseWare 00:00:17.610 --> 00:00:18.520 at ocw.mit.edu. 00:00:25.693 --> 00:00:27.110 ELIZABETH NOLAN: Last time we were 00:00:27.110 --> 00:00:33.500 working on this PKS assembly line that makes acrylide. 00:00:33.500 --> 00:00:37.580 And just as a review of that, we left off 00:00:37.580 --> 00:00:42.230 having gone over the domains and module architecture 00:00:42.230 --> 00:00:44.310 for this assembly line. 00:00:44.310 --> 00:00:48.890 So recall, each module activates a given monomer. 00:00:48.890 --> 00:00:51.740 And we can use depictions like this 00:00:51.740 --> 00:00:57.610 to show how the PKS builds a growing polyketide chain. 00:00:57.610 --> 00:01:00.740 OK, and as you saw in recitation last week, 00:01:00.740 --> 00:01:02.900 the actual structure of one of these synthases 00:01:02.900 --> 00:01:05.180 is very different than what's depicted 00:01:05.180 --> 00:01:08.510 by this left-to-right kind of assembly-line depiction there. 00:01:08.510 --> 00:01:10.910 So you saw some amazing conformational changes 00:01:10.910 --> 00:01:13.040 of the fatty acid synthase. 00:01:13.040 --> 00:01:15.410 And they're all different, but just keep that in mind 00:01:15.410 --> 00:01:16.550 when thinking about these. 00:01:16.550 --> 00:01:19.760 So this sort of notation is very helpful for us 00:01:19.760 --> 00:01:22.100 in terms of thinking about how the biosynthesis goes, 00:01:22.100 --> 00:01:26.480 but it's not an accurate representation of structure. 00:01:26.480 --> 00:01:29.210 OK, so where we left off was with looking 00:01:29.210 --> 00:01:34.370 at how these optional domains can do chemistry 00:01:34.370 --> 00:01:38.300 on the upstream monomer. 00:01:38.300 --> 00:01:42.620 And the last thing we're going to do related to this assembly 00:01:42.620 --> 00:01:46.850 line is, one, ask how is the polyketide released 00:01:46.850 --> 00:01:49.880 from the assembly line when the biosynthesis is over. 00:01:49.880 --> 00:01:52.850 And then we'll just do one exercise 00:01:52.850 --> 00:01:55.290 looking at the macrolide and working backwards. 00:01:55.290 --> 00:01:58.790 So last time, we were looking at the domain organization 00:01:58.790 --> 00:02:01.160 to determine what sort of chemistry 00:02:01.160 --> 00:02:02.900 happens to a given monomer. 00:02:02.900 --> 00:02:04.610 We can do, effectively, the opposite, 00:02:04.610 --> 00:02:07.550 looking at a natural product and identify 00:02:07.550 --> 00:02:10.370 what those monomers and properties of the assembly line 00:02:10.370 --> 00:02:11.450 are. 00:02:11.450 --> 00:02:16.610 OK, so in terms of chain release, 00:02:16.610 --> 00:02:18.335 there are thioesterase domains. 00:02:28.090 --> 00:02:32.480 And these domains are involved in chain release 00:02:32.480 --> 00:02:33.820 from the assembly line. 00:02:41.730 --> 00:02:44.660 So if you take a look in the final module 00:02:44.660 --> 00:02:49.790 here, what we see at the end is a TE for the thioesterase. 00:02:49.790 --> 00:02:56.210 OK, and so what happens in the case of DEBS is, 00:02:56.210 --> 00:03:00.830 ultimately, the chain gets transferred to a serine residue 00:03:00.830 --> 00:03:01.970 on the TE domain. 00:03:06.640 --> 00:03:11.520 OK, and I'm just going to draw the polyketide like that. 00:03:11.520 --> 00:03:15.690 And then in this case here, we remember 00:03:15.690 --> 00:03:23.790 we have the propionyl-CoA from the loading module, so 00:03:23.790 --> 00:03:25.500 the starter unit. 00:03:25.500 --> 00:03:29.310 In this case, what happens is there is a macrocyclization. 00:03:29.310 --> 00:03:31.770 So we can imagine deprotonation-- 00:03:31.770 --> 00:03:35.790 oh, excuse me, I forgot the linkage here. 00:03:35.790 --> 00:03:41.130 So for this TE, we no longer have the growing chain 00:03:41.130 --> 00:03:43.560 tethered by a Ppant arm. 00:03:43.560 --> 00:03:47.670 With the TE domain, it's tethered to the serine residue. 00:03:47.670 --> 00:03:51.900 So it's transferred from the thioester to this serine. 00:03:51.900 --> 00:03:54.405 And this here is just the polyketide in between. 00:03:57.800 --> 00:04:00.150 I'm just abbreviating it. 00:04:00.150 --> 00:04:01.560 So we can have that. 00:04:01.560 --> 00:04:04.520 We can have attack and loss. 00:04:04.520 --> 00:04:09.570 OK, so in this case what we end up with 00:04:09.570 --> 00:04:16.410 is the TE domain plus a macrocycle. 00:04:21.459 --> 00:04:27.190 And so that's how we end up with the structure as shown here. 00:04:27.190 --> 00:04:32.620 So some TE domains will result in formation of a macrocycle. 00:04:32.620 --> 00:04:35.890 Some TE domains will catalyze a hydrolytic release. 00:04:35.890 --> 00:04:37.450 And you get the linear chain. 00:04:37.450 --> 00:04:39.790 So you need to look at the natural product structure. 00:04:39.790 --> 00:04:42.760 And based on that structure, you can make an assessment 00:04:42.760 --> 00:04:46.150 as to how the TE works. 00:04:46.150 --> 00:04:49.450 And so that's also shown in this depiction and one 00:04:49.450 --> 00:04:50.750 other depiction in the notes. 00:04:50.750 --> 00:04:53.980 So here, the entire chain is drawn. 00:04:53.980 --> 00:04:56.020 And we're seeing deprotonation donation here 00:04:56.020 --> 00:04:58.690 and then attack here to give the macrocycle. 00:05:04.360 --> 00:05:13.270 So here is the product of this DEBS. 00:05:13.270 --> 00:05:17.680 And what we're going to do as a last exercise with the PKS 00:05:17.680 --> 00:05:21.190 is just look at this structure and work through identifying 00:05:21.190 --> 00:05:23.740 the monomer units and what optional domains 00:05:23.740 --> 00:05:27.430 acted on each monomer. 00:05:27.430 --> 00:05:31.960 And basically, where can we start? 00:05:31.960 --> 00:05:37.210 So if the thioesterase catalyzes a macrocyclization, 00:05:37.210 --> 00:05:42.730 that's an easy starting point, because basically, 00:05:42.730 --> 00:05:47.530 the final monomer needs to be involved there. 00:05:47.530 --> 00:05:50.200 And we know that the only place we 00:05:50.200 --> 00:05:52.390 can get a structure like this is from the starter, 00:05:52.390 --> 00:05:54.640 from that propionyl-CoA. 00:05:54.640 --> 00:05:59.320 So here, if we just look, we have the monomer 00:05:59.320 --> 00:06:02.440 from module 0, the loading. 00:06:02.440 --> 00:06:04.870 And then as we learned last time, 00:06:04.870 --> 00:06:08.770 each additional unit that gets attached to the growing 00:06:08.770 --> 00:06:12.100 polyketide gives two carbons, so two carbon units 00:06:12.100 --> 00:06:13.450 to the growing chain. 00:06:13.450 --> 00:06:18.300 So we can work our way around by two carbons, 1, 2. 00:06:18.300 --> 00:06:25.060 OK, here we have module 1, another two carbons, 00:06:25.060 --> 00:06:51.060 module 2 here, module 3, module 4, module 5, and here, 00:06:51.060 --> 00:06:51.790 module 6. 00:06:54.860 --> 00:06:57.110 So looking at a structure, you can 00:06:57.110 --> 00:06:59.900 begin to dissect what the assembly line will 00:06:59.900 --> 00:07:02.810 look like in terms of the number of modules 00:07:02.810 --> 00:07:06.290 by counting C2 units to the growing chain here. 00:07:06.290 --> 00:07:08.450 And then the other thing we can do 00:07:08.450 --> 00:07:11.450 is look at the functional group status 00:07:11.450 --> 00:07:14.030 and ask what types of optional domains 00:07:14.030 --> 00:07:18.590 needed to be there in order to give a given functional group. 00:07:18.590 --> 00:07:22.430 So for instance, in this case, in module 1 here, 00:07:22.430 --> 00:07:23.910 we're seeing an OH group. 00:07:23.910 --> 00:07:27.770 So we know there had to be the action of a keto reductase 00:07:27.770 --> 00:07:30.200 to reduce the ketone. 00:07:30.200 --> 00:07:33.410 Here we see we have this carbonyl, 00:07:33.410 --> 00:07:35.960 so there was no optional domain. 00:07:35.960 --> 00:07:37.640 In this case, what happens? 00:07:37.640 --> 00:07:38.780 We have a methylene. 00:07:38.780 --> 00:07:42.480 So that ketone we started with was fully reduced. 00:07:42.480 --> 00:07:45.270 So in this case, we have the keto reductase, 00:07:45.270 --> 00:07:51.050 the dehydratase, and the enoyl reductase. 00:07:51.050 --> 00:07:52.760 Again, here we can look at this unit. 00:07:52.760 --> 00:07:56.140 We see an OH, which tells us that there 00:07:56.140 --> 00:07:58.370 was action of a keto reductase. 00:07:58.370 --> 00:08:04.760 And here we have another OH, so we have a keto reductase. 00:08:04.760 --> 00:08:11.850 And in this case, we have none, this final one. 00:08:11.850 --> 00:08:14.540 And here, I didn't write it, but none 00:08:14.540 --> 00:08:17.840 in terms of optional domains. 00:08:17.840 --> 00:08:19.730 So this can be pretty fun. 00:08:19.730 --> 00:08:21.320 This is a pretty simple structure, 00:08:21.320 --> 00:08:23.040 but as structures get more complex, 00:08:23.040 --> 00:08:26.030 you can map out what are the optional domains there. 00:08:26.030 --> 00:08:28.970 And maybe you'll see, some other unusual structural features 00:08:28.970 --> 00:08:33.020 will indicate there is other optional domains 00:08:33.020 --> 00:08:34.471 beyond these three. 00:08:34.471 --> 00:08:35.929 And we're going to see some of that 00:08:35.929 --> 00:08:37.935 as we move into the non-ribosomal peptides. 00:08:40.710 --> 00:08:45.070 So that's given in the notes if you want to practice on that. 00:08:45.070 --> 00:08:53.510 So with this, we're going to transition into an NRPS 00:08:53.510 --> 00:08:55.700 and look at the assembly line logic 00:08:55.700 --> 00:08:58.470 for non-ribosomal peptides. 00:08:58.470 --> 00:09:03.650 And so this is a slide from last time, where we considered 00:09:03.650 --> 00:09:08.330 the starter units and extender units for fatty acids 00:09:08.330 --> 00:09:10.520 and for polyketides. 00:09:10.520 --> 00:09:13.100 And so in non-ribosomal peptides, 00:09:13.100 --> 00:09:17.510 we also have starter units and extender units, 00:09:17.510 --> 00:09:20.150 but in the case of the non-ribosomal peptide, 00:09:20.150 --> 00:09:21.640 as the name indicates, we're going 00:09:21.640 --> 00:09:25.220 to be thinking about amino acid monomers. 00:09:25.220 --> 00:09:28.250 And we're also going to be considering examples where 00:09:28.250 --> 00:09:30.380 there is aryl acid monomers. 00:09:30.380 --> 00:09:34.310 So these NRPS assembly lines will 00:09:34.310 --> 00:09:38.420 form polymers that incorporate amino acid and aryl acid 00:09:38.420 --> 00:09:41.090 monomers. 00:09:41.090 --> 00:09:43.910 And this is another slide from last time 00:09:43.910 --> 00:09:48.110 that is just summarizing the core domains and then 00:09:48.110 --> 00:09:53.300 examples of optional domains for the PKS and NRPS. 00:09:53.300 --> 00:09:58.520 So we learned last time that for PKS, every module 00:09:58.520 --> 00:10:03.170 will have a KS and a T domain, with the exception 00:10:03.170 --> 00:10:04.820 of the loading or starter module. 00:10:04.820 --> 00:10:06.590 That has no keto synthase, because there 00:10:06.590 --> 00:10:09.400 is no upstream group here. 00:10:09.400 --> 00:10:14.660 For NRPS, the core of a module is CAT trio. 00:10:14.660 --> 00:10:17.450 So condensation domain, or C domain, 00:10:17.450 --> 00:10:20.210 this is the domain that's going to catalyze peptide bond 00:10:20.210 --> 00:10:23.180 formation between two of the monomers. 00:10:23.180 --> 00:10:25.130 We have an adenylation domain. 00:10:25.130 --> 00:10:26.570 And we'll see this does chemistry 00:10:26.570 --> 00:10:30.110 similar to the aminoacyl tRNA synthetases. 00:10:30.110 --> 00:10:33.140 And then we have the T domains that 00:10:33.140 --> 00:10:37.320 are carrier proteins for the monomers and growing chain. 00:10:37.320 --> 00:10:40.430 OK, and then within a given NRPS module, 00:10:40.430 --> 00:10:42.590 there can also be optional domains. 00:10:42.590 --> 00:10:44.720 And just two examples are shown here. 00:10:44.720 --> 00:10:48.110 So maybe there is an epimerization of an amino acid. 00:10:48.110 --> 00:10:50.240 Maybe there is a methyl group and there 00:10:50.240 --> 00:10:52.660 needs to be methyltransferase to put that on. 00:10:52.660 --> 00:10:55.130 There is a lot of diversity that comes 00:10:55.130 --> 00:10:57.260 into these structures on the basis 00:10:57.260 --> 00:10:59.540 of these optional domains. 00:10:59.540 --> 00:11:01.640 And just to highlight that, I've presented 00:11:01.640 --> 00:11:05.840 here a list of possible optional domains 00:11:05.840 --> 00:11:08.495 you can find in NRPS, or for that matter, 00:11:08.495 --> 00:11:12.380 a PKS here, so all sorts of things. 00:11:12.380 --> 00:11:16.670 Look at halogenase, cyclase, reductase. 00:11:16.670 --> 00:11:21.080 There is tremendous structural diversity that can occur. 00:11:21.080 --> 00:11:29.030 OK, so if we consider the NRPS assembly line 00:11:29.030 --> 00:11:31.130 structure and notation similar to what 00:11:31.130 --> 00:11:36.170 we did with the polyketide synthases, what do we see? 00:11:49.840 --> 00:11:52.420 So I'll just draw one with two modules 00:11:52.420 --> 00:11:54.850 here, although n can indicate more. 00:11:54.850 --> 00:11:59.050 So initially what we have here is a starting or loading 00:11:59.050 --> 00:12:08.280 module, OK, so for instance, module 0. 00:12:11.520 --> 00:12:20.010 OK, here we have module 1, 2 for extenders. 00:12:24.130 --> 00:12:27.775 And here we have a thioesterase for chain release. 00:12:31.390 --> 00:12:34.000 So we'll find that in the final module 00:12:34.000 --> 00:12:39.190 like what we saw with the polyketide synthase for DEB. 00:12:39.190 --> 00:12:45.190 So this whole thing can be called an NRPS here. 00:12:47.890 --> 00:12:51.100 And what happens in terms of the action 00:12:51.100 --> 00:12:53.920 of these different core domains-- 00:12:53.920 --> 00:12:57.410 so A, we have adenylation. 00:13:06.520 --> 00:13:11.110 OK, and what these domains do are select 00:13:11.110 --> 00:13:22.160 and activate the amino acid or aryl acid monomers. 00:13:28.920 --> 00:13:31.430 OK and after these monomers are activated, 00:13:31.430 --> 00:13:34.580 the A domain also transfers them to the T domain. 00:13:37.330 --> 00:13:39.660 And we'll go over the chemistry in a minute. 00:13:45.960 --> 00:13:50.230 OK, this T domain is like what we saw with the PKS. 00:13:50.230 --> 00:14:04.550 We can call it a thiolation domain or a peptidyl carrier 00:14:04.550 --> 00:14:05.050 protein. 00:14:09.390 --> 00:14:15.840 So these T domains are going to be modified with the Ppant arm, 00:14:15.840 --> 00:14:17.845 like what we saw for PKS. 00:14:21.420 --> 00:14:25.860 We have the C domain, condensation. 00:14:35.030 --> 00:14:38.750 And so this domain capitalizes peptide bond formation. 00:14:48.890 --> 00:14:53.070 And I'll just point out here that, in contrast to the keto 00:14:53.070 --> 00:14:55.170 synthase we saw in PKS-- 00:14:55.170 --> 00:14:58.130 so we saw the keto synthase doing covalent catalysis 00:14:58.130 --> 00:15:00.350 via its cysteine residue-- 00:15:00.350 --> 00:15:03.560 the condensation domains of NRPS are involved 00:15:03.560 --> 00:15:05.230 in non-covalent catalysis. 00:15:08.720 --> 00:15:10.830 So that's just an important distinction. 00:15:14.170 --> 00:15:20.500 The growing chain does not get attached to the C domain here. 00:15:20.500 --> 00:15:23.640 And then we have the TE, so thioesterase, 00:15:23.640 --> 00:15:25.735 as we saw, for chain release. 00:15:29.130 --> 00:15:35.542 And this can be hydrolytic or macrocyclization. 00:15:45.540 --> 00:15:50.090 OK, so let's consider just the example 00:15:50.090 --> 00:15:55.760 of an NRPS that is responsible for synthesizing a tripeptide. 00:15:55.760 --> 00:15:57.145 So what is the net reaction? 00:16:13.670 --> 00:16:18.680 So imagine that we have three amino acid monomers. 00:16:22.910 --> 00:16:26.090 And I'll just point out here too that beyond knowing 00:16:26.090 --> 00:16:30.590 an epimerization domain epimerizes an amino acid, 00:16:30.590 --> 00:16:33.920 you're not responsible for stereochemistry 00:16:33.920 --> 00:16:36.320 in terms of the various structures we'll 00:16:36.320 --> 00:16:37.640 look at going through here. 00:16:37.640 --> 00:16:40.430 So I'm just not drawing stereochemistry here. 00:16:51.000 --> 00:16:52.890 So we have three amino acid monomers. 00:17:00.690 --> 00:17:04.200 There is going to be some NRPS that's 00:17:04.200 --> 00:17:06.869 responsible for formation of the tripeptide. 00:17:06.869 --> 00:17:12.569 And what we'll see is that making a trimer 00:17:12.569 --> 00:17:17.130 requires three ATPs, so one ATP per amino acid 00:17:17.130 --> 00:17:48.070 or aryl acid monomer, giving us three AMT plus three PPi here 00:17:48.070 --> 00:17:55.960 to give us our tripeptide plus three water molecules here. 00:17:55.960 --> 00:17:59.420 OK, so how does this happen? 00:17:59.420 --> 00:18:02.080 How does the NRPS take these monomers 00:18:02.080 --> 00:18:04.960 and build, say, a tripeptide? 00:18:04.960 --> 00:18:12.640 We're going to look at the ACV synthetase as a model for this. 00:18:12.640 --> 00:18:19.900 And so the ACV tripeptide is important. 00:18:19.900 --> 00:18:24.340 It forms the backbone of antibiotics of the penicillin 00:18:24.340 --> 00:18:26.140 and cephalosporin classes. 00:18:26.140 --> 00:18:29.680 So many of these are used clinically. 00:18:29.680 --> 00:18:32.920 So here are the structures of penicillin N 00:18:32.920 --> 00:18:34.810 And the cephalosporin. 00:18:34.810 --> 00:18:36.670 So at first inspection, you might not 00:18:36.670 --> 00:18:40.240 guess that these are effectively built from a tripeptide, 00:18:40.240 --> 00:18:42.820 but what happens is that a non-ribosomal peptide 00:18:42.820 --> 00:18:47.590 synthetase, the ACV synthetase, is responsible for forming 00:18:47.590 --> 00:18:50.710 two amide bonds between the three starters-- or the three 00:18:50.710 --> 00:18:51.730 monomers. 00:18:51.730 --> 00:18:53.680 And then there is additional enzymes 00:18:53.680 --> 00:18:58.990 that are responsible for modifying that peptide scaffold 00:18:58.990 --> 00:19:01.510 to give, say, this four-five fused ring 00:19:01.510 --> 00:19:06.190 system or this four-six fused ring system. 00:19:06.190 --> 00:19:10.180 OK, so what is the overall reaction of this? 00:19:10.180 --> 00:19:13.120 So similar to having these three amino acid 00:19:13.120 --> 00:19:17.770 monomers here, what we have are aminoadipate. 00:19:17.770 --> 00:19:21.240 We have L-cysteine and L-valine. 00:19:21.240 --> 00:19:24.400 And the synthetase takes these three monomers and makes 00:19:24.400 --> 00:19:26.900 this molecule here, which is called ACV. 00:19:29.430 --> 00:19:33.190 And so if we look at the synthetase in cartoon form, 00:19:33.190 --> 00:19:34.810 this is the cartoon. 00:19:34.810 --> 00:19:38.080 So we see a loading module, so just AT. 00:19:38.080 --> 00:19:41.140 Similar to the PKS, there is no catalytic domain 00:19:41.140 --> 00:19:43.750 to make a new bond in the loading module 00:19:43.750 --> 00:19:45.640 because there is nothing upstream. 00:19:45.640 --> 00:19:48.610 We see a module here, CAT. 00:19:48.610 --> 00:19:51.340 We have another CAT trio here. 00:19:51.340 --> 00:19:53.050 And then what's this? 00:19:53.050 --> 00:19:57.170 This is our first example of an optional domain within an NRPS. 00:19:57.170 --> 00:20:00.160 OK so this E is for epimerization. 00:20:00.160 --> 00:20:03.940 I mean what we'll see is that the synthetase epimerizes 00:20:03.940 --> 00:20:07.900 L-valine to D-valine during the synthesis, and then 00:20:07.900 --> 00:20:09.670 the thioesterase. 00:20:09.670 --> 00:20:13.270 So similar to what we did with the PKS, for the NRPS, 00:20:13.270 --> 00:20:18.040 you can count T domains as a way to identify the modules 00:20:18.040 --> 00:20:20.815 and to figure out how many monomers are involved. 00:20:25.310 --> 00:20:29.390 I also just point out-- and this builds upon Colin's comment 00:20:29.390 --> 00:20:30.910 from last time-- 00:20:30.910 --> 00:20:34.280 is that this assembly line is responsible 00:20:34.280 --> 00:20:36.610 only for the synthesis of a tripeptide, 00:20:36.610 --> 00:20:38.300 but look at its size. 00:20:38.300 --> 00:20:41.270 It's greater than 450 kilodaltons. 00:20:41.270 --> 00:20:46.520 That's quite big-- so a large enzyme, 10 different domains, 00:20:46.520 --> 00:20:50.000 all just for synthesis of this one tripeptide here. 00:20:53.210 --> 00:20:54.350 So what happens? 00:21:03.580 --> 00:21:09.240 We're going to go over the action of the A domains 00:21:09.240 --> 00:21:13.170 and the T domains first. 00:21:13.170 --> 00:21:15.600 And then we'll look at a cartoon in the slides. 00:21:22.370 --> 00:21:25.330 So the first points to make are that we 00:21:25.330 --> 00:21:30.800 need to have loading of the assembly line. 00:21:30.800 --> 00:21:34.480 So amino acids need to be selected and activated. 00:21:37.380 --> 00:21:40.420 And that's where these A domains come in. 00:21:40.420 --> 00:21:41.710 So what's happening? 00:21:45.570 --> 00:21:50.390 So we have some amino acid monomers-- 00:21:50.390 --> 00:21:54.650 so maybe it's the L-cysteine, for instance, or the L-valine-- 00:21:54.650 --> 00:21:57.710 plus ATP. 00:21:57.710 --> 00:22:00.650 The A domain does chemistry similar to what 00:22:00.650 --> 00:22:05.270 we saw with the aminoacyl tRNA synthetases 00:22:05.270 --> 00:22:08.108 to form an activated intermediate. 00:22:14.090 --> 00:22:17.150 So we get an amino adenylate here. 00:22:17.150 --> 00:22:18.140 And then what happens? 00:22:18.140 --> 00:22:19.085 So the T domain-- 00:22:24.120 --> 00:22:34.500 OK, and this T domain must be modified by a PPTase, 00:22:34.500 --> 00:22:45.630 like what we saw for the PKS, to have the Ppant arm. 00:22:49.550 --> 00:22:52.370 After we have activation of the amino acid 00:22:52.370 --> 00:22:54.800 or aryl acid monomer, the A domain 00:22:54.800 --> 00:22:58.220 is going to assist with transfer of this monomer to the Ppant 00:22:58.220 --> 00:22:59.830 arm of the T domain here. 00:23:24.500 --> 00:23:33.040 OK, so we got an aminoacyl-S-T covalently tethered 00:23:33.040 --> 00:23:37.240 via a thioester linkage. 00:23:37.240 --> 00:23:42.880 So one ATP is consumed per monomer loaded. 00:23:42.880 --> 00:23:47.020 And the ATP PPi exchange assay we discussed back 00:23:47.020 --> 00:23:50.450 in the translation module for studying the aminoacyl tRNA 00:23:50.450 --> 00:23:56.560 synthetases is used all the time to study new A domains 00:23:56.560 --> 00:23:58.930 and ask what amino acid or aryl acid 00:23:58.930 --> 00:24:02.470 monomers do they activate here. 00:24:02.470 --> 00:24:05.390 So that assay comes up in this type of work. 00:24:08.290 --> 00:24:17.700 So what happens then in terms of formation of a peptide bond, 00:24:17.700 --> 00:24:26.315 we're going to consider condensation by the C domains. 00:24:33.040 --> 00:24:35.330 And so let's just imagine-- 00:24:35.330 --> 00:24:37.970 we're just going to draw two modules. 00:24:37.970 --> 00:24:47.290 So we have a loading module and then a first extender module. 00:24:47.290 --> 00:24:55.160 And the T domains have been post translationally modified 00:24:55.160 --> 00:24:56.750 with the Ppant arm. 00:24:56.750 --> 00:24:59.962 And the action of the A domains has loaded the amino acids 00:24:59.962 --> 00:25:00.545 at this stage. 00:25:06.310 --> 00:25:10.180 OK, so we have some amino acid loaded here. 00:25:10.180 --> 00:25:19.320 And then we have some amino acid loaded here. 00:25:21.920 --> 00:25:23.980 OK, and what happens? 00:25:23.980 --> 00:25:26.280 We're going to have nucleophilic attack 00:25:26.280 --> 00:25:30.900 from the alpha amino group onto the upstream monomer 00:25:30.900 --> 00:25:32.345 and then transfer of this monomer. 00:25:35.740 --> 00:25:38.455 And this occurs via the action of the C domain. 00:25:58.560 --> 00:26:01.200 We have R2. 00:26:01.200 --> 00:26:09.280 And now we have formation of our new peptide bond. 00:26:09.280 --> 00:26:16.780 Sorry, this is R2 here. 00:26:16.780 --> 00:26:20.290 And as I noted above, there is no covalent catalysis 00:26:20.290 --> 00:26:22.240 with the C domain. 00:26:22.240 --> 00:26:25.230 Somehow it's helping to bring these chains together 00:26:25.230 --> 00:26:28.300 and to allow this nucleophilic attack to occur 00:26:28.300 --> 00:26:30.550 and to allow the monomer to be transferred, 00:26:30.550 --> 00:26:32.680 but this unit is never transferred 00:26:32.680 --> 00:26:33.870 to the C domain itself. 00:26:33.870 --> 00:26:34.370 Yeah? 00:26:34.370 --> 00:26:36.880 AUDIENCE: So is the C domain responsible for deprotonating 00:26:36.880 --> 00:26:37.570 the NH2? 00:26:37.570 --> 00:26:39.790 Or is that just always-- 00:26:39.790 --> 00:26:41.230 ELIZABETH NOLAN: Yeah, I don't-- 00:26:41.230 --> 00:26:43.610 how this gets deprotonated, I don't know. 00:26:43.610 --> 00:26:45.340 But this is back to similar, like what 00:26:45.340 --> 00:26:47.230 we saw in the ribosome. 00:26:47.230 --> 00:26:50.650 And somehow, this alpha amino group needs to be deprotonated. 00:26:50.650 --> 00:26:52.840 And there is something in the environment 00:26:52.840 --> 00:26:55.660 of this machine that's allowing that to happen, 00:26:55.660 --> 00:26:58.610 but whether it's the C domain or something else, 00:26:58.610 --> 00:27:00.580 yeah, I don't know the answer to that. 00:27:03.350 --> 00:27:08.890 So let's look at a cartoon of this with this ACV synthase. 00:27:08.890 --> 00:27:12.970 So here we have on top the synthase 00:27:12.970 --> 00:27:17.290 loaded with the amino acid monomers. 00:27:17.290 --> 00:27:22.480 OK, so we see loading module and then two extender modules. 00:27:22.480 --> 00:27:24.850 We have the aminoadipate. 00:27:24.850 --> 00:27:28.630 So it's not a canonical amino acid, but it's amino-acid-like. 00:27:28.630 --> 00:27:32.260 We have the cysteine and the valine. 00:27:32.260 --> 00:27:35.860 What happens as these condensation reactions occur, 00:27:35.860 --> 00:27:37.600 we get chain elongation. 00:27:37.600 --> 00:27:40.660 So this is depicted here in a similar manner 00:27:40.660 --> 00:27:43.210 to how that PKS assembly line was depicted. 00:27:46.630 --> 00:27:52.540 So formation of two peptide bonds, and then what happens? 00:27:52.540 --> 00:27:55.390 Ultimately, we have chain transfer 00:27:55.390 --> 00:27:59.320 to a serine residue on the thioesterase domain. 00:27:59.320 --> 00:28:02.420 And this is a case where the thioesterase domain catalyzes 00:28:02.420 --> 00:28:04.510 this hydrolytic release. 00:28:04.510 --> 00:28:06.400 So as opposed to macrocyclization, 00:28:06.400 --> 00:28:09.040 we're seeing activation of a water molecule 00:28:09.040 --> 00:28:13.800 and attack, which releases this ACV tripeptide. 00:28:13.800 --> 00:28:16.570 OK, and I've drawn the ACV tripeptide here 00:28:16.570 --> 00:28:22.330 to indicate effectively getting to this structure. 00:28:22.330 --> 00:28:26.020 So what happens after this tripeptide is released 00:28:26.020 --> 00:28:28.810 from the assembly line, is that there is additional enzymes 00:28:28.810 --> 00:28:30.850 that play a tailoring role. 00:28:30.850 --> 00:28:33.370 So like, for proteins we talk about post-translational 00:28:33.370 --> 00:28:36.550 modification, for these types of natural products, 00:28:36.550 --> 00:28:39.760 we talk about post-assembly-line tailoring. 00:28:39.760 --> 00:28:44.760 And so in this case, there is some enzymes such as IPNF, 00:28:44.760 --> 00:28:47.920 and non-heme iron enzyme that's responsible 00:28:47.920 --> 00:28:53.170 for oxygenated cyclization to give the fused ring system 00:28:53.170 --> 00:28:59.250 characteristics of these beta-lactams like isopenicillin 00:28:59.250 --> 00:29:00.760 N. 00:29:00.760 --> 00:29:04.360 We can look at this in another cartoon form. 00:29:04.360 --> 00:29:07.750 So here is the holoform. 00:29:07.750 --> 00:29:10.720 Recall, we called the T domains apo 00:29:10.720 --> 00:29:13.930 when the serine is not post-translationally modified 00:29:13.930 --> 00:29:15.550 with the Ppant arm. 00:29:15.550 --> 00:29:18.730 And the T domains are holo when the Ppant 00:29:18.730 --> 00:29:22.630 arm has been attached, as indicated by this squiggle. 00:29:22.630 --> 00:29:26.620 We then have loading of the amino acid monomers 00:29:26.620 --> 00:29:29.470 via the action of the A domains. 00:29:29.470 --> 00:29:33.410 So formation of that aminoacyl AMP 00:29:33.410 --> 00:29:39.460 or amino adenylate intermediate, so one monomer per module. 00:29:39.460 --> 00:29:43.240 We have chain elongation events catalyzed by the condensation 00:29:43.240 --> 00:29:44.950 domain. 00:29:44.950 --> 00:29:50.200 We have chain transfer to the TE domain as shown here, 00:29:50.200 --> 00:29:56.490 chain transfer, and then chain release here, 00:29:56.490 --> 00:29:58.440 and then post-assembly-line tailoring. 00:30:01.350 --> 00:30:04.380 So with that in mind, what we're going to do now 00:30:04.380 --> 00:30:09.120 is look at another non-ribosomal peptide synthetase. 00:30:09.120 --> 00:30:12.810 This one synthesizes the backbone 00:30:12.810 --> 00:30:16.170 of the antibiotic vancomycin. 00:30:16.170 --> 00:30:22.110 And the structure of vancomycin is shown here. 00:30:22.110 --> 00:30:24.690 This is an antibiotic that's basically 00:30:24.690 --> 00:30:28.530 considered one of last resort for bacterial infections. 00:30:28.530 --> 00:30:31.860 And there is a huge problem of vancomycin resistance 00:30:31.860 --> 00:30:34.320 in the clinic these days. 00:30:34.320 --> 00:30:37.320 So at first glance, this molecule 00:30:37.320 --> 00:30:40.170 might not look like it's based on a peptide. 00:30:40.170 --> 00:30:41.670 But then if you look more carefully, 00:30:41.670 --> 00:30:44.000 you see there is a lot of amide bonds. 00:30:44.000 --> 00:30:47.880 And there is also some other things going in 00:30:47.880 --> 00:30:50.250 to get this final structure. 00:30:50.250 --> 00:30:54.390 So effectively, the backbone of vancomycin 00:30:54.390 --> 00:30:59.220 is a polypeptide that's a sevenmer. 00:30:59.220 --> 00:31:02.640 So within this heptapeptide scaffold, 00:31:02.640 --> 00:31:05.370 there are two proteinogenic amino acids 00:31:05.370 --> 00:31:10.170 and five non-proteinogenic amino acids here. 00:31:10.170 --> 00:31:15.750 And because we have seven amino-acid-type monomers, 00:31:15.750 --> 00:31:18.810 we need an assembly line that has seven modules, one 00:31:18.810 --> 00:31:23.010 module per amino acid monomer. 00:31:23.010 --> 00:31:27.780 And what we'll see is that these seven modules are distributed 00:31:27.780 --> 00:31:32.200 over three proteins. 00:31:32.200 --> 00:31:36.960 We have a case of a thioesterase catalyzing hydrolytic release. 00:31:36.960 --> 00:31:38.760 And then we're going to need to think 00:31:38.760 --> 00:31:41.520 about what are the other tailoring enzymes involved 00:31:41.520 --> 00:31:44.200 in giving vancomycin this structure. 00:31:44.200 --> 00:31:46.230 So for instance, look here. 00:31:46.230 --> 00:31:49.800 We see there is this aryl-aryl C-C bond. 00:31:49.800 --> 00:31:52.710 We see these aryl-ether connections. 00:31:52.710 --> 00:31:55.590 And we also have these sugars attached. 00:31:55.590 --> 00:31:57.480 And look, there is also an N-methylation 00:31:57.480 --> 00:32:02.220 here of leucine 1, so a lot happening. 00:32:02.220 --> 00:32:06.510 And the consequence of this post-assembly-line tailoring 00:32:06.510 --> 00:32:09.990 is that, what's a linear sevenmer polypeptide ends up 00:32:09.990 --> 00:32:13.860 having an architecture that's described as a dome, so 00:32:13.860 --> 00:32:15.750 a dome-shaped architecture. 00:32:15.750 --> 00:32:20.340 And what vancomycin does is that it blocks biosynthesis 00:32:20.340 --> 00:32:24.390 of the bacterial cell wall by binding to a certain lipid 00:32:24.390 --> 00:32:26.760 precursor in that. 00:32:26.760 --> 00:32:30.360 So let's look at the assembly line. 00:32:30.360 --> 00:32:33.210 And this is just an overview of the tailoring 00:32:33.210 --> 00:32:35.040 I just told you about. 00:32:35.040 --> 00:32:37.440 And this is the amino acid sequence 00:32:37.440 --> 00:32:39.510 in order of the different monomers there 00:32:39.510 --> 00:32:44.290 and the identities of the non-proteinogenic amino acids. 00:32:44.290 --> 00:32:48.040 So here is the assembly line. 00:32:48.040 --> 00:32:53.190 And if we take a look, we have the loading module, AT. 00:32:53.190 --> 00:32:59.340 We can count the T domains to give us the modules involved 00:32:59.340 --> 00:33:00.210 in extension. 00:33:00.210 --> 00:33:02.280 So there is seven T domains. 00:33:02.280 --> 00:33:05.460 And look, CAT, CAT, CAT-- 00:33:05.460 --> 00:33:09.180 we have a number of optional epimerization domains. 00:33:09.180 --> 00:33:12.750 And at the end, we see this TE domain. 00:33:12.750 --> 00:33:17.130 And so you can walk through and look at each monomer being 00:33:17.130 --> 00:33:19.890 attached to the growing chain. 00:33:19.890 --> 00:33:21.750 And then what do we see? 00:33:21.750 --> 00:33:24.510 What we see happening down here is 00:33:24.510 --> 00:33:29.820 that when we have the linear polypeptide attached 00:33:29.820 --> 00:33:32.610 to this module here, what happens 00:33:32.610 --> 00:33:35.730 is that there is some tailoring happening 00:33:35.730 --> 00:33:39.870 while the polypeptide is still attached to the assembly line. 00:33:39.870 --> 00:33:43.510 So enzymes that are not parts of the assembly line 00:33:43.510 --> 00:33:46.720 but are involved in the biosynthesis can come in. 00:33:46.720 --> 00:33:49.890 And sometimes they'll modify the chain 00:33:49.890 --> 00:33:53.460 when it's still attached to the NRPS or PKS. 00:33:53.460 --> 00:33:55.230 Or sometimes they do the chemistry 00:33:55.230 --> 00:33:57.840 after the chain is released. 00:33:57.840 --> 00:34:00.810 And often, this is a question that people need 00:34:00.810 --> 00:34:02.390 to sort out experimentally. 00:34:02.390 --> 00:34:04.785 So in this case here, we see that there 00:34:04.785 --> 00:34:08.730 is some oxidative cross-linking that occurs while the chain is 00:34:08.730 --> 00:34:10.409 still attached to the T domain. 00:34:10.409 --> 00:34:12.780 So there is formation of the aryl-ether bond 00:34:12.780 --> 00:34:15.460 and this aryl-aryl bond here. 00:34:15.460 --> 00:34:17.190 And then after the chain is released 00:34:17.190 --> 00:34:20.190 in a hydrolytic manner, what happens 00:34:20.190 --> 00:34:24.059 is the sugars get attached post-assembly-line here. 00:34:24.059 --> 00:34:25.139 Do you have a question? 00:34:25.139 --> 00:34:27.030 AUDIENCE: Yeah, are the enzymes ever 00:34:27.030 --> 00:34:29.690 actually in the assembly line, like the optional domains 00:34:29.690 --> 00:34:30.190 of PKS? 00:34:30.190 --> 00:34:32.370 Or in this case, is it always such 00:34:32.370 --> 00:34:34.260 that the enzymes are separate? 00:34:34.260 --> 00:34:36.570 ELIZABETH NOLAN: It will depend on the assembly line. 00:34:36.570 --> 00:34:37.987 Yeah, so that's something you need 00:34:37.987 --> 00:34:42.719 to look for in the assembly line from the bioinformatics. 00:34:42.719 --> 00:34:45.690 So in this case, we're only seeing epimerization domains 00:34:45.690 --> 00:34:48.270 in the assembly line, but there can easily 00:34:48.270 --> 00:34:50.550 be methyltransferases, or reductases, 00:34:50.550 --> 00:34:54.150 or cyclases-- any number of possibilities 00:34:54.150 --> 00:34:57.810 within the assembly line itself there. 00:34:57.810 --> 00:35:03.080 And these optional domains will work on the upstream monomer. 00:35:03.080 --> 00:35:07.130 This is just an example of the tailoring enzymes involved 00:35:07.130 --> 00:35:10.790 for cross-linking of this vancomycin scaffold. 00:35:10.790 --> 00:35:15.560 In this case, there are three cytochrome P450 enzymes 00:35:15.560 --> 00:35:23.360 that are needed in order to make these cross-links. 00:35:23.360 --> 00:35:26.210 And that chemistry is shown here to get 00:35:26.210 --> 00:35:29.330 to what's called the vancomycin aglycone, which 00:35:29.330 --> 00:35:34.040 means that there are no sugars attached. 00:35:34.040 --> 00:35:36.260 And I won't draw this one on the board, 00:35:36.260 --> 00:35:38.960 but you can do a similar exercise 00:35:38.960 --> 00:35:41.150 with this molecule or any others in terms 00:35:41.150 --> 00:35:44.060 of identifying the monomer units from the structure 00:35:44.060 --> 00:35:45.740 for yourself. 00:35:45.740 --> 00:35:48.980 So if we're looking here, we have 00:35:48.980 --> 00:35:52.730 effectively the N-terminus, so the starter, 00:35:52.730 --> 00:35:56.270 and then effectively look at the peptide bonds 00:35:56.270 --> 00:35:59.900 and work your way through to find the different monomers 00:35:59.900 --> 00:36:00.590 here. 00:36:00.590 --> 00:36:02.930 So by doing that, if you're given a natural product, 00:36:02.930 --> 00:36:05.150 you can figure out how many modules are 00:36:05.150 --> 00:36:07.160 needed in the assembly line. 00:36:07.160 --> 00:36:08.960 And you can also make an assessment 00:36:08.960 --> 00:36:12.680 as to what other types of chemistry might have to happen. 00:36:12.680 --> 00:36:15.440 And I'll just keep in mind, for something like this-- 00:36:15.440 --> 00:36:18.410 let's just take this for an example with this halogen. 00:36:18.410 --> 00:36:21.410 You might ask, well, is that part of the monomer? 00:36:21.410 --> 00:36:24.800 Or is that atom incorporated sometime down the road? 00:36:24.800 --> 00:36:26.450 OK, those are types of questions people 00:36:26.450 --> 00:36:31.140 who explore biosynthesis of these molecules think about. 00:36:31.140 --> 00:36:36.920 OK, so with that in mind, let's take a look at some examples. 00:36:36.920 --> 00:36:41.720 And the questions are, what kind of assembly line is this? 00:36:41.720 --> 00:36:43.240 How many monomers? 00:36:43.240 --> 00:36:47.880 And maybe there will be some extra questions as we go. 00:36:47.880 --> 00:36:50.000 So here we have an assembly line that's 00:36:50.000 --> 00:36:53.490 required to make an antibiotic called daptomycin. 00:36:53.490 --> 00:36:55.580 And a company down the street in Lexington 00:36:55.580 --> 00:36:59.570 called Cubist has done a lot of work on this natural product. 00:36:59.570 --> 00:37:01.130 So how many monomers are here? 00:37:09.324 --> 00:37:13.270 Yeah, 13, right-- so count these T domains 00:37:13.270 --> 00:37:15.280 based on what's seen here. 00:37:15.280 --> 00:37:16.675 How many optional domains? 00:37:19.900 --> 00:37:21.763 AUDIENCE: Three. 00:37:21.763 --> 00:37:23.680 ELIZABETH NOLAN: And then what else do we see? 00:37:23.680 --> 00:37:26.650 So we see that this assembly line 00:37:26.650 --> 00:37:32.980 is divided over three proteins, effectively, here. 00:37:32.980 --> 00:37:35.140 And similar to what we saw with DEBS, 00:37:35.140 --> 00:37:37.900 when we have a break in the cartoon, 00:37:37.900 --> 00:37:41.260 that indicates a new polypeptide chain. 00:37:41.260 --> 00:37:41.980 What's missing? 00:37:48.693 --> 00:37:49.735 AUDIENCE: Loading module. 00:37:49.735 --> 00:37:52.550 ELIZABETH NOLAN: Yeah, there is no loading module here, 00:37:52.550 --> 00:37:55.710 right, no AT at the beginning. 00:37:55.710 --> 00:37:56.850 So what's going on? 00:38:02.600 --> 00:38:05.370 So in this case, I haven't shown you a structure. 00:38:05.370 --> 00:38:08.880 It highlights there is always exceptions to the rule. 00:38:08.880 --> 00:38:12.150 What happens here is that the loading module actually 00:38:12.150 --> 00:38:18.390 loads a fatty acid, so not a standard monomer for NRPS. 00:38:18.390 --> 00:38:21.100 So that fatty acid has to come from somewhere. 00:38:21.100 --> 00:38:22.740 And you can think about discussions 00:38:22.740 --> 00:38:26.280 here as to where that may have come from. 00:38:26.280 --> 00:38:31.580 Look at how big this is, 624 kilodaltons, 783, 256-- 00:38:31.580 --> 00:38:34.150 we're on the order of 1.5 megadaltons. 00:38:34.150 --> 00:38:37.455 This is huge for a 13-mer natural product. 00:38:40.710 --> 00:38:43.410 What about this one? 00:38:43.410 --> 00:38:44.490 What do we see here? 00:38:49.630 --> 00:38:51.070 So this is a natural product-- 00:38:51.070 --> 00:38:55.150 this makes the natural product produced by Streptomyces 00:38:55.150 --> 00:38:57.850 that has insecticidal activity. 00:38:57.850 --> 00:38:59.320 And it kills parasitic worms. 00:38:59.320 --> 00:39:03.610 But anyhow, what kind of natural product 00:39:03.610 --> 00:39:05.380 is produced by this assembly line? 00:39:08.380 --> 00:39:10.480 We have a polyketide, right? 00:39:10.480 --> 00:39:11.335 How many modules? 00:39:28.328 --> 00:39:30.065 [INAUDIBLE] the T domains. 00:39:30.065 --> 00:39:31.190 AUDIENCE: [INAUDIBLE] 00:39:31.190 --> 00:39:32.900 ELIZABETH NOLAN: Yeah, 13 again, right-- 00:39:32.900 --> 00:39:36.950 four proteins, 13 modules, so how many 00:39:36.950 --> 00:39:38.225 unmodified beta ketones? 00:39:41.715 --> 00:39:46.329 What would you want to look for for a modified beta ketone? 00:39:46.329 --> 00:39:47.810 AUDIENCE: [INAUDIBLE] 00:39:47.810 --> 00:39:50.520 ELIZABETH NOLAN: Exactly, no optional domains-- so how many 00:39:50.520 --> 00:39:52.350 of those? 00:39:52.350 --> 00:39:56.040 Yeah, right, so two modules, we have one 00:39:56.040 --> 00:40:01.635 here and then one over here with no optional domains. 00:40:05.250 --> 00:40:06.150 What about this one? 00:40:09.980 --> 00:40:11.730 This is for a molecule called bleomycin. 00:40:11.730 --> 00:40:17.600 JoAnne is an expert on the mechanism of this molecule. 00:40:17.600 --> 00:40:18.300 What's going on? 00:40:35.415 --> 00:40:36.810 OK, there is a lot going on. 00:40:36.810 --> 00:40:38.160 This one is very complicated. 00:40:38.160 --> 00:40:40.530 But in terms of making an assessment 00:40:40.530 --> 00:40:44.988 about the type of biosynthetic logic, what do we see here? 00:40:44.988 --> 00:40:47.360 AUDIENCE: [INAUDIBLE] 00:40:47.360 --> 00:40:49.020 ELIZABETH NOLAN: Right, so what we 00:40:49.020 --> 00:40:52.290 see is that there is both non-ribosomal peptide 00:40:52.290 --> 00:40:54.830 synthesis happening and polyketide 00:40:54.830 --> 00:40:58.230 biosynthesis happening in this assembly line. 00:40:58.230 --> 00:41:01.020 And that tells us that the product metabolite 00:41:01.020 --> 00:41:03.750 is a PKS-NRPS hybrid. 00:41:03.750 --> 00:41:05.410 OK, so what do we see? 00:41:05.410 --> 00:41:07.740 We see all of these CAT trios which 00:41:07.740 --> 00:41:10.740 are indicative of non-ribosomal peptide biosynthesis. 00:41:10.740 --> 00:41:12.630 And then what's happening here? 00:41:12.630 --> 00:41:16.470 We have a module that's using polyketide machinery. 00:41:16.470 --> 00:41:20.070 And then we go back to non-ribosomal-peptide-based 00:41:20.070 --> 00:41:20.870 logic here. 00:41:23.440 --> 00:41:24.960 We have many proteins, right? 00:41:24.960 --> 00:41:28.800 So this assembly line is divided over many proteins. 00:41:28.800 --> 00:41:32.100 And look, we see that even some of the modules are divided up. 00:41:32.100 --> 00:41:35.100 So for instance, this CAT trio is 00:41:35.100 --> 00:41:38.080 divided between two proteins. 00:41:38.080 --> 00:41:43.209 So you may not have all domains of a module on a given protein. 00:41:43.209 --> 00:41:45.630 AUDIENCE: What happens if you have two C domains in a row? 00:41:45.630 --> 00:41:48.130 ELIZABETH NOLAN: So where do you see two C domains in a row? 00:41:48.130 --> 00:41:51.548 AUDIENCE: Between BlmV and BlmX. 00:41:51.548 --> 00:41:53.024 ELIZABETH NOLAN: Five and-- 00:41:53.024 --> 00:41:55.405 AUDIENCE: Is that actually in a row? 00:41:55.405 --> 00:41:57.530 ELIZABETH NOLAN: Yeah, so then that's the question. 00:41:57.530 --> 00:42:00.390 Are they actually in a row? 00:42:00.390 --> 00:42:04.072 AUDIENCE: Further down, four Cy cyclases without any C domain. 00:42:04.072 --> 00:42:05.780 ELIZABETH NOLAN: Yeah, so that's actually 00:42:05.780 --> 00:42:06.738 where I was going next. 00:42:06.738 --> 00:42:12.800 So what's going on with the Cy without a C domain? 00:42:12.800 --> 00:42:15.950 So what's happening-- and we'll probably, if there is time, 00:42:15.950 --> 00:42:19.580 go over an example of this on Friday-- 00:42:19.580 --> 00:42:24.350 is that Cy, so these cyclization domains 00:42:24.350 --> 00:42:27.480 are a variant on a condensation domain. 00:42:27.480 --> 00:42:30.050 And what they do is, they both catalyze formation 00:42:30.050 --> 00:42:33.440 of the peptide bond and then they catalyze-- after that, 00:42:33.440 --> 00:42:36.350 they catalyze formation of a heterocycle. 00:42:36.350 --> 00:42:38.210 So if you recall, I believe we looked 00:42:38.210 --> 00:42:40.430 at the structure of yersiniabactin 00:42:40.430 --> 00:42:44.240 during the first lecture on these. 00:42:44.240 --> 00:42:45.890 It has a number of heterocycles. 00:42:45.890 --> 00:42:49.490 And those form by this Cy domain. 00:42:49.490 --> 00:42:51.730 And we can see that here in the structure. 00:42:51.730 --> 00:42:53.870 So what I've done on this slide is 00:42:53.870 --> 00:42:57.040 just present to you the structures, 00:42:57.040 --> 00:42:58.820 so the natural products that result 00:42:58.820 --> 00:43:00.890 from these different assembly lines. 00:43:00.890 --> 00:43:05.510 And if we take a look at the bleomycin, what do we see here? 00:43:05.510 --> 00:43:09.480 We have these two heterocycles that are fused together. 00:43:09.480 --> 00:43:15.290 And those are formed via the action of these two cyclization 00:43:15.290 --> 00:43:16.980 domains down here. 00:43:16.980 --> 00:43:21.020 So effectively, these originate from cysteine. 00:43:21.020 --> 00:43:24.110 So cysteines, and serines, and threonines 00:43:24.110 --> 00:43:29.180 can end up forming structures like these if there is 00:43:29.180 --> 00:43:31.250 the appropriate type of domain. 00:43:31.250 --> 00:43:34.490 This molecule is extremely complicated here. 00:43:34.490 --> 00:43:36.650 And so it's a good puzzle to look at it 00:43:36.650 --> 00:43:41.210 and try to sort out what are the monomers in it in here. 00:43:41.210 --> 00:43:46.316 Does anyone know what this does, bleomycin? 00:43:46.316 --> 00:43:48.272 AUDIENCE: [INAUDIBLE] 00:43:51.700 --> 00:43:56.040 ELIZABETH NOLAN: Well, so it's an anticancer antibiotic here. 00:43:56.040 --> 00:43:58.080 It can intercalate into DNA. 00:43:58.080 --> 00:44:00.900 And these heterocycles are important for that. 00:44:00.900 --> 00:44:03.233 And then it causes strand breaks. 00:44:03.233 --> 00:44:04.650 And I've actually learned recently 00:44:04.650 --> 00:44:06.450 it's also used for, like, treating arts. 00:44:06.450 --> 00:44:09.680 So it will kill HPV that causes warts. 00:44:09.680 --> 00:44:14.470 Anyhow, all of these compounds have interesting activities, 00:44:14.470 --> 00:44:18.600 which is one reason why they can be of interest. 00:44:18.600 --> 00:44:24.450 So with the logic in place, where we're going to close 00:44:24.450 --> 00:44:29.670 this module is thinking about how folks study these in lab. 00:44:29.670 --> 00:44:33.720 So say you want to figure out the biosynthesis of a molecule 00:44:33.720 --> 00:44:36.810 like daptomycin or bleomycin, what 00:44:36.810 --> 00:44:39.660 is it that one needs to do? 00:44:39.660 --> 00:44:42.030 And something just to keep in mind with this right 00:44:42.030 --> 00:44:44.610 off the bat, is that these are huge. 00:44:44.610 --> 00:44:46.560 So some of these examples here, if you 00:44:46.560 --> 00:44:50.850 take a look at the sizes, they're, like, comparable 00:44:50.850 --> 00:44:53.830 to the prokaryotic ribosome. 00:44:53.830 --> 00:44:56.550 That's a huge protein assembly. 00:44:56.550 --> 00:44:59.640 And that presents a limitation from the standpoint 00:44:59.640 --> 00:45:04.020 of doing experimental work, because trying 00:45:04.020 --> 00:45:08.670 to overexpress or produce these assembly lines in something 00:45:08.670 --> 00:45:11.850 like E. coli is typically just unreasonable. 00:45:11.850 --> 00:45:14.610 And in terms of a native producer organism, 00:45:14.610 --> 00:45:16.740 say, something like Streptomyces, 00:45:16.740 --> 00:45:18.630 we may or may not know conditions 00:45:18.630 --> 00:45:22.080 that cause the organism to make the natural product, so 00:45:22.080 --> 00:45:24.920 conditions that cause it to express this machinery, 00:45:24.920 --> 00:45:26.950 and then even if it made at a-- 00:45:26.950 --> 00:45:29.220 in an amount that's useful. 00:45:29.220 --> 00:45:33.000 So what happens? 00:45:33.000 --> 00:45:35.190 What are we going to do as experimentalists? 00:45:35.190 --> 00:45:39.870 So as I said, we need to keep in mind that these machines are 00:45:39.870 --> 00:45:41.350 enormous. 00:45:41.350 --> 00:45:43.230 And so we need to take this into account 00:45:43.230 --> 00:45:46.980 during experimental design. 00:45:46.980 --> 00:45:52.260 And these days, bioinformatics drives a lot of the studies. 00:45:52.260 --> 00:45:54.697 So rather than first finding a natural product 00:45:54.697 --> 00:45:56.280 and determining its structure and then 00:45:56.280 --> 00:45:59.700 hunting down the protein machinery, a wealth of genomes 00:45:59.700 --> 00:46:01.020 are becoming available. 00:46:01.020 --> 00:46:04.140 And so you can use bioinformatics to search 00:46:04.140 --> 00:46:08.430 for PKS or NRPS gene clusters. 00:46:08.430 --> 00:46:11.880 And then you can make some assessment 00:46:11.880 --> 00:46:15.120 as to what type of molecule these gene clusters might 00:46:15.120 --> 00:46:16.860 be responsible for making. 00:46:16.860 --> 00:46:20.370 So bioinformatics plays a huge role. 00:46:20.370 --> 00:46:24.450 And it allows us to predict the domains, 00:46:24.450 --> 00:46:28.980 to predict their locations, and predict their boundaries here. 00:46:28.980 --> 00:46:32.370 So as I just said, overexpression 00:46:32.370 --> 00:46:35.700 of a complete assembly line is generally not feasible. 00:46:35.700 --> 00:46:37.440 So what do people do? 00:46:37.440 --> 00:46:43.770 People will typically express individual domains or maybe 00:46:43.770 --> 00:46:48.930 di-domains and study those in the test tube. 00:46:48.930 --> 00:46:54.150 So you can imagine PCR amplifying an A domain or a T 00:46:54.150 --> 00:46:58.800 domain, or maybe the A and T domain together, 00:46:58.800 --> 00:47:01.170 and then creating some plasmid that allows 00:47:01.170 --> 00:47:04.980 you to express that in E. coli. 00:47:04.980 --> 00:47:07.230 So there is a lot of overexpression. 00:47:07.230 --> 00:47:09.060 The proteins need to be purified, 00:47:09.060 --> 00:47:11.820 so maybe something like affinity chromatography that we've 00:47:11.820 --> 00:47:13.710 spoken about before. 00:47:13.710 --> 00:47:16.170 And then a key point is that, in order 00:47:16.170 --> 00:47:18.570 to have any of this chemistry work, 00:47:18.570 --> 00:47:22.590 these T domains need to be post-translationally modified 00:47:22.590 --> 00:47:24.660 by the Ppant arm. 00:47:24.660 --> 00:47:27.710 And if you're overexpressing a T domain from Streptomyces 00:47:27.710 --> 00:47:30.750 or some organism in E. coli, you can pretty much 00:47:30.750 --> 00:47:34.330 assume there is no PPTase in E. coli 00:47:34.330 --> 00:47:36.990 that's going to do this for you. 00:47:36.990 --> 00:47:39.810 So you need to do that after the fact. 00:47:39.810 --> 00:47:45.720 And so there needs to be a PPTase. 00:47:45.720 --> 00:47:49.470 And what we'll see is that there is 00:47:49.470 --> 00:47:54.320 a PPTase from B. subtilis called SFP that's very promiscuous. 00:47:54.320 --> 00:47:57.330 It will basically modify any T domain. 00:47:57.330 --> 00:48:00.540 And so experimentally, this is what people use, 00:48:00.540 --> 00:48:06.450 because often, one has no clue what the endogenous PPTase is 00:48:06.450 --> 00:48:10.530 here, so SFP to the rescue. 00:48:10.530 --> 00:48:12.870 In terms of activity assay, so once 00:48:12.870 --> 00:48:16.440 you have your domains or di-domains purified, 00:48:16.440 --> 00:48:18.210 what happens? 00:48:18.210 --> 00:48:20.640 This is the typical flow. 00:48:20.640 --> 00:48:24.780 So the first is to characterize the A domains and to ask, 00:48:24.780 --> 00:48:29.260 what amino acid or aryl acid is activated by the A domain 00:48:29.260 --> 00:48:31.140 and what is the selectivity? 00:48:31.140 --> 00:48:32.880 And by getting that information, you 00:48:32.880 --> 00:48:35.970 have a good clue as to what monomer a given 00:48:35.970 --> 00:48:38.580 module is responsible for. 00:48:38.580 --> 00:48:41.970 And the ATP-PPi exchange assay we discussed 00:48:41.970 --> 00:48:45.090 in the context of the aminoacyl tRNA synthetases 00:48:45.090 --> 00:48:47.100 is commonly employed. 00:48:47.100 --> 00:48:50.280 So this is where we use the radiolabeled ATP 00:48:50.280 --> 00:48:53.020 and took into reversibility there. 00:48:53.020 --> 00:48:56.680 So go back and review that assay as needed. 00:48:56.680 --> 00:48:59.890 There will be some examples of this in the problem set. 00:48:59.890 --> 00:49:04.180 So once the A domain activity is known 00:49:04.180 --> 00:49:07.850 in terms of preferred monomer, the next question is, 00:49:07.850 --> 00:49:12.820 will that A domain transfer the amino acid monomer 00:49:12.820 --> 00:49:14.650 to a given T domain? 00:49:14.650 --> 00:49:18.820 So you design assays to look for transfer of the activated 00:49:18.820 --> 00:49:22.330 monomer to the post-translationally-modified T 00:49:22.330 --> 00:49:23.950 domain here. 00:49:23.950 --> 00:49:25.960 So in these assays, there is a lot 00:49:25.960 --> 00:49:31.630 of work with radiolabels, with HPLC, and mass spec. 00:49:31.630 --> 00:49:35.560 So once these T domains are loaded, 00:49:35.560 --> 00:49:38.500 you can look for peptide bond formation. 00:49:38.500 --> 00:49:43.360 So imagine you have an isolated T domain from a loading module 00:49:43.360 --> 00:49:47.380 that you've stuck the amino acid on and then you have this guy, 00:49:47.380 --> 00:49:49.690 the next question is, does the C domain 00:49:49.690 --> 00:49:53.320 catalyze bond formation reaction? 00:49:53.320 --> 00:49:56.640 And again, we'll see there is a lot of use of radiolabels, 00:49:56.640 --> 00:50:01.990 HPLC, SDS-PAGE here. 00:50:01.990 --> 00:50:05.980 And then you know, there is the question of the TE domain 00:50:05.980 --> 00:50:09.250 and the TE domain catalyzing chain release. 00:50:09.250 --> 00:50:12.520 So it's quite systematic in terms of how you work through 00:50:12.520 --> 00:50:19.360 from identifying an assembly line to then teasing apart 00:50:19.360 --> 00:50:21.970 the various activities of the different domains 00:50:21.970 --> 00:50:23.530 and different modules. 00:50:23.530 --> 00:50:27.430 And so where we'll close this module on Friday 00:50:27.430 --> 00:50:32.140 is with looking at the experiments that 00:50:32.140 --> 00:50:35.980 were done for the biosynthesis of an iron chelator produced 00:50:35.980 --> 00:50:42.550 by E. coli and working through basically you know, how was it 00:50:42.550 --> 00:50:47.500 that this NRPS was found? 00:50:47.500 --> 00:50:50.050 What were the experiments done to identify 00:50:50.050 --> 00:50:52.330 the different activities of the different domains? 00:50:52.330 --> 00:50:55.900 And it's really that work that has 00:50:55.900 --> 00:50:58.210 served as a foundation and a paradigm 00:50:58.210 --> 00:51:03.700 for many, many further studies of these systems here. 00:51:03.700 --> 00:51:05.530 And so with that, we'll close for today. 00:51:05.530 --> 00:51:09.570 And there is no class Wednesday, so I'll see you on Friday.