Biochemical Analysis of the Substrate Specificity of the β-Ketoacyl-Acyl Carrier Protein Synthase Domain of Module 2 of the Erythromycin Polyketide Synthase

Wu, Joy T.; Kinoshita, Kenji; Khosla, Chaitan; Cane, David E.

doi:10.1021/bi048147g

Cited by 48 publications

(47 citation statements)

References 43 publications

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“…This hypothesis would be consistent with the well-established mechanism for chain length control by iterative (type II) PKSs (28). Alternatively, the tetraketide may be back-transferred to KS3, but fail to undergo another round of chain elongation, also observed in prior studies on DEBS module 2 in which di-and triketide substrates could be loaded onto the KS domain but could not undergo subsequent chain elongation (29). Notably, such an abortive back-transfer would be expected to result in eventual inactivation of the KS3 domain by the deadend intermediate.…”

Section: Resultssupporting

confidence: 86%

Reprogramming a module of the 6-deoxyerythronolide B synthase for iterative chain elongation

Kapur¹,

Lowry

Yuzawa³

et al. 2012

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

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101

103

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Multimodular polyketide synthases (PKSs) have an assembly line architecture in which a set of protein domains, known as a module, participates in one round of polyketide chain elongation and associated chemical modifications, after which the growing chain is translocated to the next PKS module. The ability to rationally reprogram these assembly lines to enable efficient synthesis of new polyketide antibiotics has been a long-standing goal in natural products biosynthesis. We have identified a ratchet mechanism that can explain the observed unidirectional translocation of the growing polyketide chain along the 6-deoxyerythronolide B synthase. As a test of this model, module 3 of the 6-deoxyerythronolide B synthase has been reengineered to catalyze two successive rounds of chain elongation. Our results suggest that high selectivity has been evolutionarily programmed at three types of protein-protein interfaces that are present repetitively along naturally occurring PKS assembly lines.A fundamental challenge to our understanding of multimodular polyketide synthases (PKSs) is the ability to explain the unidirectional translocation of growing polyketide chains through these enzymatic assembly lines. The 6-deoxyerythronolide B synthase (DEBS; Fig. 1) is arguably the most well studied example of assembly line PKSs (1-4). Each module of DEBS has an acyl carrier protein (ACP) that collaborates with the β-ketosynthase (KS) domain of the same module to catalyze a single round of polyketide chain elongation ( Fig. 2). At this point, the ACP-bound intermediate is precluded from back-transfer to the same KS domain and is instead translocated to the KS domain of the downstream module (Fig. 2).In the course of our investigations into the mechanism of intermodular chain translocation (Fig. 2, reaction 1) and intramodular chain elongation (Fig. 2, reaction 4) within DEBS, we discovered that the specificity of these two reactions is controlled by protein-protein interfaces involving distinct regions of the ACP domain (5). In the present study, we have used site-directed mutagenesis to identify ACP residues that contribute to the observed specificity. In turn, these residue-level constraints were exploited to validate the proposed structural model for ACP docking during chain elongation (5), as well as to develop an analogous in silico model for chain translocation. Generalization of these models to three naturally occurring PKSs has revealed a programming pattern that establishes a ratchet mechanism that accurately explains the unique chain translocation pathway of each PKS. As a test of this ratcheting mechanism in PKS assembly lines, we engineered a module of DEBS to iteratively catalyze two successive rounds of chain elongation instead of only one. Results and DiscussionIdentification of ACP Residues That Contribute to Chain Elongation Specificity. All ACPs from fatty acid and polyketide synthases are all-helical bundles comprised of three major α-helices connected by two structured loops (Fig. 3A). In earlier work (5) we showe...

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Section: Resultssupporting

confidence: 86%

Reprogramming a module of the 6-deoxyerythronolide B synthase for iterative chain elongation

Kapur¹,

Lowry

Yuzawa³

et al. 2012

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

Self Cite

101

103

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“…Thus, acyl transfer between an ACP and the immediate downstream KS may be difficult to control, leading to relatively limited enzymatic selectivity, and a potentially reversible process. Indeed, it has been observed that a large excess of SNAC thiol is able to remove an acyl group from the KS active site by transthioesterification [22] .…”

Section: Acyl Chain Elongation Drives Ketosynthase Substrate Selectivmentioning

confidence: 99%

Acyl‐Chain Elongation Drives Ketosynthase Substrate Selectivity in trans‐Acyltransferase Polyketide Synthases

et al. 2014

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“…Besides the active-site Cys (36), each KS domain harbors a pair of universally conserved His residues (for example, His 334 and His 374 in DEBS KS5) that facilitate the decarboxylation and condensation reactions (37,38). (2S,3R)-2-Methyl-3-hydroxypentanoyl-N-acetylcysteamine thioester (NDK-SNAc), the N-acetylcysteamine acyl thioester analog of the natural ACP-bound syn-diketide substrate of DEBS module 2, can conveniently be used as a surrogate substrate for DEBS KS domains (39,40).…”

Section: Ks Domainsmentioning

confidence: 99%

Programming of Erythromycin Biosynthesis by a Modular Polyketide Synthase

Cane

2010

Journal of Biological Chemistry

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Since the discovery and sequencing of 6-deoxyerythronolide B synthase 20 years ago, this exceptionally large, multifunctional protein remains the paradigm for the understanding of the structure and biochemical function of modular polyketide synthases. The broad-spectrum macrolide antibiotic erythromycin is one of several hundred closely related, branched chain, polyoxygenated polyketides, many of which are widely used in human and veterinary medicine as antibiotic, immunosuppressant, antitumor, antifungal, and antiparasitic agents. The multistep assembly of the parent macrocyclic aglycon, 6-deoxyerythronolide B (6-dEB), 2 is controlled by a large (2 MDa) modular protein known as 6-dEB synthase (DEBS) (1-4). The biosynthetic intermediates never leave the polyketide synthase (PKS) but are passed along the DEBS assembly line from one acyl carrier protein (ACP) domain to the next. In fact, DEBS has served as the prototype of modular PKS gene clusters, dozens of which of both known and unknown function have now been sequenced from bacterial sources (5-7).Each homodimeric DEBS subunit contains two 160 -200-kDa protein modules, each responsible for a single round of polyketide chain extension and functional group modification. Within each module are several catalytic domains of 100 -400 amino acids each that are analogous in structure, function, and organization to the corresponding fatty acid synthase (FAS) components (8 -10). All six DEBS modules contain three core domains consisting of 1) a ␤-ketoacyl-ACP synthase (the ketosynthase (KS) domain) that catalyzes the key polyketide chainbuilding reaction, a decarboxylative condensation of a methylmalonyl-ACP building block with the polyketide chain provided by the upstream PKS module (see Figs. 1 and 2); 2) an acyltransferase (AT) domain that specifically loads the methylmalonyl-CoA extender unit onto the flexible 18-Å phosphopantetheine arm of the ACP domain; and 3) the ACP domain itself, which carries the polyketide biosynthetic intermediates from domain to domain and then delivers the resulting product to the KS domain of the downstream module. Additional FASlike domains are responsible for modification of the oxidation state and stereochemistry of the growing ACP-bound intermediates: a ␤-ketoacyl-ACP reductase (KR) domain, a dehydratase (DH) domain, and an enoyl reductase (ER) domain. At the N terminus of the most upstream module is a loading didomain that primes the KS domain of module 1 with the propionyl starter unit. Finally, cyclization of the full-length macrocyclic polyketide and release of 6-dEB are controlled by a dedicated thioesterase domain located at the C terminus of the most downstream module. Programming of Polyketide BiosynthesisThe chain length, substitution pattern, and oxidation level of the initially generated, full-length heptaketide 6-dEB are the direct consequence of the number of DEBS modules as well as the domain composition of each module (Fig. 1) (8 -11). The striking colinearity between the organization of the constituent biosynthetic do...

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Biochemical Analysis of the Substrate Specificity of the β-Ketoacyl-Acyl Carrier Protein Synthase Domain of Module 2 of the Erythromycin Polyketide Synthase

Cited by 48 publications

References 43 publications

Reprogramming a module of the 6-deoxyerythronolide B synthase for iterative chain elongation

Reprogramming a module of the 6-deoxyerythronolide B synthase for iterative chain elongation

Acyl‐Chain Elongation Drives Ketosynthase Substrate Selectivity in trans‐Acyltransferase Polyketide Synthases

Programming of Erythromycin Biosynthesis by a Modular Polyketide Synthase

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