Reconstituting Modular Activity from Separated Domains of 6-Deoxyerythronolide B Synthase

Kim, Chu‐Young; Alekseyev, Viktor Y.; Chen, Alice Y.; Tang, Yong; Cane, David E.; Khosla, Chaitan

doi:10.1021/bi048418n

Cited by 68 publications

(101 citation statements)

References 25 publications

(35 reference statements)

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“…Although the EntB-ArCP mutants we studied were found to be significantly deficient in their ability to interact with EntF, M249A and A268Q could still be efficiently acylated by the free-standing (13,14,37). As precedent, it appears that the eukaryotic protein ubiquitin, about the same size as carrier protein domains, is also an information-rich scaffold, with at least two surfaces that can be recognized and differentiated by ubiquitin-binding proteins to direct the many distinct outcomes initiated by ubiquitylation of proteins (38,39).…”

Section: Discussionmentioning

confidence: 88%

A protein interaction surface in nonribosomal peptide synthesis mapped by combinatorial mutagenesis and selection

Lai

Fischbach

Liu

et al. 2006

Proc. Natl. Acad. Sci. U.S.A.

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Nonribosomal peptide synthetases (NRPSs) and polyketide synthases are large, multidomain enzymes that biosynthesize a number of pharmaceutically important natural products. The recognition of biosynthetic intermediates, displayed via covalent attachment to carrier proteins, by catalytic domains is critical for NRPS and polyketide synthase function. We report the use of combinatorial mutagenesis coupled with in vivo selection for the production of the Escherichia coli NRPS product enterobactin to map the surface of the aryl carrier protein (ArCP) domain of EntB that interacts with the downstream elongation module EntF. Two libraries spanning the predicted helix 2 and loop 2͞helix 3 of EntB-ArCP were generated by shotgun alanine scanning and selected for their ability to support enterobactin production. From the surviving pools, we identified several hydrophobic residues (M249, F264, and A268) that were highly conserved. These residues cluster near the phosphopantetheinylated serine in a structural model, and two of these positions are in the predicted helix 3 region. Subsequent in vitro studies are consistent with the hypothesis that these residues form a surface on EntB required for interaction with EntF. These results suggest that helix 3 is a major recognition element in EntB-ArCP and demonstrate the utility of selection-based approaches for studying NRPS biosynthesis. nonribosomal peptide synthetase ͉ siderophore A number of medicinally useful natural products are biosynthesized in their cognate producer organisms by nonribosomal peptide synthetase (NRPS) and polyketide synthase (PKS) mutidomain enzymes (1). Examples of NRPS-and PKS-derived compounds include the antibiotics erythromycin (PKS) and vancomycin (NRPS) (2, 3), the antitumor agents epothilone and bleomycin (both from hybrid PKS͞NRPS systems) (4, 5), and the immunosuppressant rapamycin (hybrid PKS͞NRPS) (6). At various points during the biosynthesis of these molecules, substrates for catalytic operations are presented as thioesters tethered to carrier proteins through a 4Ј-phosphopantetheine cofactor. These carrier proteins, peptidyl carrier proteins (PCPs) in NRPSs and acyl carrier proteins (ACPs) in PKSs, are homologous to the ACPs that are involved in production of fatty acids from primary metabolism (7). ACPs and PCPs are small (Ϸ80-100 residues) and contain a central serine residue that is the site of attachment for the phosphopantetheine arm (1, 7). Carrier proteins thus provide the protein scaffolding for display of the growing acyl chains in each module.Critical to the function of NRPSs and PKSs are the interactions between domains (8). Because the biosynthetic intermediates are covalently tethered to carrier proteins, studying the determinants of carrier-protein recognition by other synthetase domains remains an important goal. In a typical cycle of NRPS elongation (that is, formation of an amide bond between two amino acids), a PCP must be recognized by the adenylation domain, which acylates the phosphopantetheine with an amino acyl group ...

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Section: Discussionmentioning

confidence: 88%

A protein interaction surface in nonribosomal peptide synthesis mapped by combinatorial mutagenesis and selection

Lai

Fischbach

Liu

et al. 2006

Proc. Natl. Acad. Sci. U.S.A.

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“…S2A). These structurally characterized truncated modules included KS and AT domains together with both flanking and intervening linker sequences but lacked the KR and ACP domains of the parent DEBS modules (9,10,20). Although this assay involves several elementary steps (for details, see Fig.…”

Section: Resultsmentioning

confidence: 99%

“…(In the chain elongation reaction, a nucleophilic malonyl extender unit is attached to the ACP, whereas the growing polyketide chain itself is attached to the ACP via an electrophilic thioester linkage when it is ready for forward transfer.) Previous studies, however, suggest that protein-protein recognition between the KS and the ACP domains also plays an important role in both reactions (7)(8)(9)(10). Moreover, this protein-protein recognition not only influences the specificity (k cat ∕K M ) of each reaction, but also the maximum rate constant (k cat ).…”

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confidence: 99%

Molecular recognition between ketosynthase and acyl carrier protein domains of the 6-deoxyerythronolide B synthase

Kapur

Chen²,

Cane³

et al. 2010

Proc. Natl. Acad. Sci. U.S.A.

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115

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Every polyketide synthase module has an acyl carrier protein (ACP) and a ketosynthase (KS) domain that collaborate to catalyze chain elongation. The same ACP then engages the KS domain of the next module to facilitate chain transfer. Understanding the mechanism for this orderly progress of the growing polyketide chain represents a fundamental challenge in assembly line enzymology. Using both experimental and computational approaches, the molecular basis for KS-ACP interactions in the 6-deoxyerythronolide B synthase has been decoded. Surprisingly, KS-ACP recognition is controlled at different interfaces during chain elongation versus chain transfer. In fact, chain elongation is controlled at a docking site remote from the catalytic center. Not only do our findings reveal a new principle in the modular control of polyketide antibiotic biosynthesis, they also provide a rationale for the mandatory homodimeric structure of polyketide synthases, in contrast to the monomeric nonribosomal peptide synthetases.assembly line enzymes | polyketide synthase | protein-protein interactions M odular polyketide synthases such as 6-deoxyerythronolide B synthase (DEBS) are assembly lines of catalytic modules responsible for the biosynthesis of polyketide natural products (1-6). A fundamental challenge toward elucidating their molecular logic is to understand the mechanism by which covalently bound biosynthetic intermediates are sequentially accessed by individual catalytic sites of the synthase. Most notably, each module of a polyketide synthase (PKS) has an acyl carrier protein (ACP) domain that collaborates with the β-ketosynthase (KS) domain of the same module to catalyze polyketide chain elongation, and subsequently engages with the KS domain of the next module to facilitate forward chain transfer (Fig. 1). The simplest explanation for the orderly progress of a growing polyketide chain along a multimodular PKS is that each ACP is equivalently recognized by both KS partners, and that selection of the appropriate KS-ACP pair at a given point in the catalytic cycle is solely dictated by the identity of the substrate anchored on the ACP. (In the chain elongation reaction, a nucleophilic malonyl extender unit is attached to the ACP, whereas the growing polyketide chain itself is attached to the ACP via an electrophilic thioester linkage when it is ready for forward transfer.) Previous studies, however, suggest that protein-protein recognition between the KS and the ACP domains also plays an important role in both reactions (7-10). Moreover, this protein-protein recognition not only influences the specificity (k cat ∕K M ) of each reaction, but also the maximum rate constant (k cat ).In the course of our efforts to elucidate the principles that govern KS-ACP recognition during chain elongation and chain transfer, we have made two unexpected and potentially important observations. First, notwithstanding the fact that the same active sites are deployed in both reactions, the structural elements that mediate KS-ACP recognition are entir...

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“…By reconstituting chimeric PKS systems from combinations of heterologous domains, it has become possible to systematically probe the substrate specificity and stereochemistry of a variety of PKS domains. 6 . We have now established the stereochemistry of each of these reductions and shed light on the diastereoselectivity of each ketoreductase, using a sensitive method that can in principle be extended to any recombinant KR domain capable of reducing an ACP-bound 3-ketoacyl-ACP triketide.…”

Section: Discussionmentioning

confidence: 99%

Stereospecificity of Ketoreductase Domains of the 6-Deoxyerythronolide B Synthase

Castonguay

Chen

et al. 2007

J. Am. Chem. Soc.

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Abstract6-Deoxyerythronolide B synthase (DEBS) is a modular polyketide synthase (PKS) responsible for the biosynthesis of 6-dEB (1), the parent aglycone of the broad spectrum macrolide antibiotic erythromycin. Individual DEBS modules, which contain the catalytic domains necessary for each step of polyketide chain elongation and chemical modification, can be deconstructed into constituent domains. To better understand the intrinsic stereospecificity of the ketoreductase (KR) domains, an in vitro reconstituted system has been developed involving combinations of ketosynthase (KS) -acyl transferase (AT) didomains with acyl-carrier protein (ACP) and KR domains from different DEBS modules. Incubations with (2S,3R)-2-methyl-3-hydroxypentanoic acid N-acetylcysteamine thioester (2) and methylmalonyl-CoA plus NADPH result in formation of a reduced, ACP-bound triketide that is converted to the corresponding triketide lactone 4 by either base-or enzyme-catalyzed hydrolysis/cyclization. A sensitive and robust GC-mass spectrometry technique has been developed to assign the stereochemistry of the resulting triketide lactones, on the basis of direct comparison with synthetic standards of each of the four possible diasteromers 4a-4d. Using the [KS][AT] didomains from either DEBS module 3 or module 6 in combination with KR domains from modules 2 or 6 gave in all cases exclusively (2R,3S,4R,5R)-3,5-dihydroxy-2,4-dimethyl-n-heptanoic acid-δ-lactone (4a). The same product was also generated by a chimeric module in which [KS3][AT3] was fused to [KR5][ACP5] and the DEBS thioesterase [TE] domain. Reductive quenching of the ACPbound 2-methyl-3-ketoacyl triketide intermediate with sodium borohydride confirmed that in each case the triketide intermediate carried only an unepimerized D-2-methyl group. The results confirm the predicted stereospecificity of the individual KR domains, while revealing an unexpected configurational stability of the ACP-bound 2-methyl-3-ketoacyl thioester intermediate. The methodology should be applicable to the study of any combination of heterologous [KS][AT] and [KR] domains.Modular polyketide synthases (PKSs) are multifunctional enzymes responsible for the biosynthesis of polyketides metabolites that exhibit a wide range of important biological properties, including antibacterial, antifungal, anticancer, and immunosuppressive activity. 1 They catalyze repetitive Claisen-like condensations between methylmalonyl or malonyl thioester building blocks and the growing polyketide acyl thioesters. Using an assembly line of active sites, each module is responsible for one cycle of polyketide chain elongation and *To whom correspondence should be addressed. E-mail: David_Cane@brown.edu. (TE) domain, located at the C-terminus of the furthest downstream module, catalyzes release and concomitant cyclization of the mature, full-length macrocyclic polyketide. NIH Public AccessThe 6-deoxyerythronolide synthase (DEBS) from Saccharopolyspora erythraea, which is by far the most thoroughly studied modular PKS, is responsib...

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Reconstituting Modular Activity from Separated Domains of 6-Deoxyerythronolide B Synthase

Cited by 68 publications

References 25 publications

A protein interaction surface in nonribosomal peptide synthesis mapped by combinatorial mutagenesis and selection

A protein interaction surface in nonribosomal peptide synthesis mapped by combinatorial mutagenesis and selection

Molecular recognition between ketosynthase and acyl carrier protein domains of the 6-deoxyerythronolide B synthase

Stereospecificity of Ketoreductase Domains of the 6-Deoxyerythronolide B Synthase

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