Dissecting and Exploiting Intermodular Communication in Polyketide Synthases

Gokhale, Rajesh S.; Tsuji, Stuart Y.; Cane, David E.; Khosla, Chaitan

doi:10.1126/science.284.5413.482

Cited by 320 publications

(329 citation statements)

References 19 publications

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“…15 For the expression of the hybrid module containing KS3, AT3, KR5, ACP5, and TE from DEBS, denoted M3M5TE, pAYC46 was constructed using the gene SOEing method. 25 First round PCR was used to produce two fragments, one encoding module 3 from the natural BsiWI site to the Tyr-Arg junction site (using primers 5′ -GCCATATGGTGGCGCTCGTACGGCGTGGAGCCC -3′ and 5′ -GCTTCCCGCCATACGACGCGGTAGGCCAGCTCG -3′), and another encoding module 5 from the Val-Val-Trp junction site to the natural BbvCI site with an EcoRI site added (using primers 5′ -CGAGCTGGCCTACCGCGTCGTATGGCGGGAAGC -3′ and 5′ -GCGAATTCAGCTCCTCCCTCAGCTCGGACGCGC -3′).…”

Section: Expression and Purification Of Debs Module 3 + Te (M3te) Andmentioning

confidence: 99%

Extender Unit and Acyl Carrier Protein Specificity of Ketosynthase Domains of the 6-Deoxyerythronolide B Synthase

et al. 2006

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Polyketide synthases (PKSs) catalyze the production of numerous biologically important natural products via repeated decarboxylative condensation reactions. Modular PKSs, such as the 6-deoxyerythronolide B synthase (DEBS), consist of multiple catalytic modules, each containing a unique set of covalently linked catalytic domains. To better understand the engineering opportunities of these assembly lines, the extender unit and acyl carrier protein (ACP) specificity of ketosynthase (KS) domains from modules 3 and 6 of DEBS were analyzed. These studies were undertaken with a newly developed didomain [KS][AT] construct, which lacks its own ACP domain and can therefore be interrogated with homologous or heterologous ACP or acyl-ACP substrates. By substituting the natural methylmalonyl extender unit with a malonyl group, a modest role was demonstrated for the KS in recognition of the nucleophilic substrate. The KS domain from module 3 of DEBS was found to exhibit a distinct ACP-recognition profile from the KS domain of module 6. Based on the above kinetic insights, a hybrid module was constructed ([TE]) which displayed substrate recognition and elongation capabilities consistent with the natural module 3 protein. Unlike module 3 however, which lacks a ketoreductase (KR) domain, the hybrid module was able to catalyze reduction of the β-ketothioester product of chain elongation. The high expression level and functionality of this hybrid protein demonstrates the usefulness of kinetic analysis for hybrid module design.

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Section: Expression and Purification Of Debs Module 3 + Te (M3te) Andmentioning

confidence: 99%

Extender Unit and Acyl Carrier Protein Specificity of Ketosynthase Domains of the 6-Deoxyerythronolide B Synthase

et al. 2006

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“…Early investigations had revealed that selective protein-protein interfaces located outside the core catalytic modules play important roles in channeling intermediates through a PKS assembly line. 6,7 (Such interfaces are designated as shape-complementary tabs in Fig. 1.)…”

Section: Introductionmentioning

confidence: 99%

Probing the interactions of an acyl carrier protein domain from the 6‐deoxyerythronolide B synthase

et al. 2011

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The assembly-line architecture of polyketide synthases (PKSs) provides an opportunity to rationally reprogram polyketide biosynthetic pathways to produce novel antibiotics. A fundamental challenge toward this goal is to identify the factors that control the unidirectional channeling of reactive biosynthetic intermediates through these enzymatic assembly lines. Within the catalytic cycle of every PKS module, the acyl carrier protein (ACP) first collaborates with the ketosynthase (KS) domain of the paired subunit in its own homodimeric module so as to elongate the growing polyketide chain and then with the KS domain of the next module to translocate the newly elongated polyketide chain. Using NMR spectroscopy, we investigated the features of a structurally characterized ACP domain of the 6-deoxyerythronolide B synthase that contribute to its association with its KS translocation partner. Not only were we able to visualize selective protein-protein interactions between the two partners, but also we detected a significant influence of the acyl chain substrate on this interaction. A novel reagent, CF 3 -S-ACP, was developed as a 19 F NMR spectroscopic probe of protein-protein interactions. The implications of our findings for understanding intermodular chain translocation are discussed.

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“…4 For example, DEBS module 3+TE has been shown to catalyze the conversion of (2S,3R)-2-methyl-3-hydroxypentanoic acid N-acetylcysteamine thioester (diketide-SNAC 2) and methylmalonyl-CoA to the triketide ketolactone 3 which is generated by TE-catalyzed release of the corresponding acyclic 2,4-dimethyl-3-keto-5-hydroxypentanoyl thioester from the ACP (Figure 2A). 5 In similar fashion, DEBS module 2 +TE, module 5+TE, and module 6+TE, each carrying an active KR domain, all convert diketide 2 and methylmalonyl-CoA to the reduced triketide 3-hydroxylactone 4a ( Figure 2B). 5 Sitedirected mutagenesis of specific domains and swapping of entire domains between different DEBS modules as well as with heterologous PKS modules has further clarified the role of individual PKS domains, while allowing the generation of novel, rationally engineered polyketides.…”

mentioning

confidence: 96%

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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Dissecting and Exploiting Intermodular Communication in Polyketide Synthases

Cited by 320 publications

References 19 publications

Extender Unit and Acyl Carrier Protein Specificity of Ketosynthase Domains of the 6-Deoxyerythronolide B Synthase

Extender Unit and Acyl Carrier Protein Specificity of Ketosynthase Domains of the 6-Deoxyerythronolide B Synthase

Probing the interactions of an acyl carrier protein domain from the 6‐deoxyerythronolide B synthase

Stereospecificity of Ketoreductase Domains of the 6-Deoxyerythronolide B Synthase

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