Stereospecificity of Ketoreductase Domains of the 6-Deoxyerythronolide B Synthase

Castonguay, Roselyne; He, Weiguo; Chen, Alice Y.; Khosla, Chaitan; Cane, David E.

doi:10.1021/ja0753290

Cited by 81 publications

(94 citation statements)

References 51 publications

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“…Recombinant DEBS KR1 reduces racemic 2-methyl-3-ketopentanoyl-SNAc exclusively to a syn-(2S,3R)-2-methyl-3-hydroxy diketide (NDK-SNAc), thereby demonstrating that this reductase is completely specific not only for reduction on the re-face of the ketone carbonyl but also for the natural L-configuration of the adjacent 2-methyl group, consistent with the overall stereospecificity of DEBS module 1 (59,60 (Fig. 4a) (61,62). DEBS KR2, KR5, and KR6 are completely diastereoselective, generating a product with the identical (2R,3S)-2-methyl-3-hydroxy stereochemistry as that of the parent DEBS modules from which they had been derived ( Figs.…”

Section: Stereochemistry Of Kr-catalyzed 3-ketoacyl-acp Reductionssupporting

confidence: 57%

“…DEBS KR2, KR5, and KR6 are completely diastereoselective, generating a product with the identical (2R,3S)-2-methyl-3-hydroxy stereochemistry as that of the parent DEBS modules from which they had been derived ( Figs. 1 and 4a (61,62). Unexpectedly, this ACP-bound 2-methyl-3-ketoacyl triketide thioester was configurationally remarkably stable, undergoing Ͻ5-15% epimerization even after 1 h, Ͼ15-45-fold slower than the measured rate for buffer-catalyzed deuterium exchange of untethered methyl-2-methyl acetoacetate.…”

Section: Stereochemistry Of Kr-catalyzed 3-ketoacyl-acp Reductionsmentioning

confidence: 82%

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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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Section: Stereochemistry Of Kr-catalyzed 3-ketoacyl-acp Reductionssupporting

confidence: 57%

Section: Stereochemistry Of Kr-catalyzed 3-ketoacyl-acp Reductionsmentioning

confidence: 82%

Programming of Erythromycin Biosynthesis by a Modular Polyketide Synthase

Cane

2010

Journal of Biological Chemistry

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“…Alkaline hydrolysis of 14 C-labeled DHm was attempted to identify the possible thioester intermediate on enzyme (21,26,27). Release of 14 C-labeled 6MSA from the labeled DHm was detected by TLC autoradiography analysis (Fig.…”

Section: Resultsmentioning

confidence: 99%

Hidden Function of Catalytic Domain in 6-Methylsalicylic Acid Synthase for Product Release

Moriguchi

Kezuka

Nonaka

et al. 2010

Journal of Biological Chemistry

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Functional investigation of the proposed dehydratase domain of ATX, a 6-methylsalicylic acid synthase from Aspergillus terreus, revealed that the domain is not involved in dehydration of the ␤-hydroxytriketide intermediate tethered on the acyl carrier protein but catalyzes thioester hydrolysis to release the product from the acyl carrier protein. Thus, we renamed this domain the thioester hydrolase (TH) domain. The intermediate bound to the TH domain of mutant H972A formed in the presence of NADPH was released as 6-methylsalicylic acid by both the intact ATX and by THID (a 541-amino acid region containing TH domain and its downstream) protein, in trans. Furthermore, THID showed a catalytic activity to hydrolyze a model substrate, 6-methylsalicylic acid-N-acetylcysteamine. The TH domain is the first example of a product-releasing domain that is located in the middle of a multidomain iterative type I polyketide synthase. Moreover, it is functionally different from serine protease-type thioesterase domains of iterative type I polyketide synthases.Iterative type I polyketide synthases (iPKSs) 3 are large multifunctional enzymes consisting of the minimum catalytic domains for PKS reactions, the ␤-ketoacyl synthase (KS), acyl transferase (AT) and acyl carrier protein (ACP) domains, and additional domains such as a ketoreductase (KR), dehydratase (DH), enoylreductase (ER), methyltransferase, and thioesterase (TE) domain. Through iterative use of these domains in a programmed manner, each iPKS produces its specific polyketide product. However, little is known how iPKSs control their reactions (1).6-Methylsalicylic acid synthase (MSAS) was the first PKS to be purified and the first fungal iPKS whose gene was cloned (2-6). A conserved domain search indicated that MSAS consists of KS, AT, DH, KR, and ACP domains (5, 7). These domains of MSAS are thought to catalyze a series of programmed reactions, Claisen condensations, reduction, dehydration, cyclization, and product release (Fig. 1).Because MSAS is one of the simplest iPKSs, we have been studying its catalytic mechanism using ATX cloned from Aspergillus terreus (8). We previously carried out the expression of catalytic domain mutants of ATX in yeast. The KS mutant (KSm), AT domain mutant (ATm), and ACP mutant (ACPm) lost 6-methylsalicylic acid (6MSA) production, and the KR domain mutant (KRm) produced triacetic acid lactone as reported (9).The DH domain of ATX was assigned by comparison of secondary structural elements predicted by PredictProtein with discrete DHs of type II fatty acid synthase (FAS) from Escherichia coli (10). The conserved DH domain motif, HXXXGXXXXP, has been identified in FAS DH, in which the histidine residue is a catalytic residue for dehydration, (11) and the corresponding sequence H 972 XXXGXXXXP 981 was found in ATX. This His 972 is crucial for ATX reaction as indicated by our previous result that the ATX H972A mutant (DHm) did not give any product, such as the ␤-hydroxy triketide (9).Because MSAS has no apparent TE domain, it has been one of...

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“…7A). Furthermore, in a reconstituted system containing recombinant KR2, the ketoreductase domain derived from DEBS module 2, and added NADPH, the initially generated ACP3-bound 3-ketoacyl triketide is diastereospecifically reduced to yield an ACP3-bound intermediate that is identical in structure and stereochemistry to the natural substrate of DEBS module 3 (24). Notwithstanding this identity, the wild-type ACP3 does not appreciably back-transfer the attached triketide to KS3 (Fig.…”

Section: A Model For Unidirectional Chain Translocation In Multimodulmentioning

confidence: 99%

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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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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Stereospecificity of Ketoreductase Domains of the 6-Deoxyerythronolide B Synthase

Cited by 81 publications

References 51 publications

Programming of Erythromycin Biosynthesis by a Modular Polyketide Synthase

Programming of Erythromycin Biosynthesis by a Modular Polyketide Synthase

Hidden Function of Catalytic Domain in 6-Methylsalicylic Acid Synthase for Product Release

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

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