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Title: Extender unit and acyl carrier protein specificity of ketosynthase domains of the 6-deoxyerythronolide B synthase
Authors: Chen, A.Y.
Schnarr, N.A.
Kim, C.-Y. 
Cane, D.E.
Khosla, C.
Issue Date: 8-Mar-2006
Citation: Chen, A.Y., Schnarr, N.A., Kim, C.-Y., Cane, D.E., Khosla, C. (2006-03-08). Extender unit and acyl carrier protein specificity of ketosynthase domains of the 6-deoxyerythronolide B synthase. Journal of the American Chemical Society 128 (9) : 3067-3074. ScholarBank@NUS Repository.
Abstract: 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 keto synthase (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. On the basis of the above kinetic insights, a hybrid module was constructed ([KS3][AT3][KR5][ACP5][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. © 2006 American Chemical Society.
Source Title: Journal of the American Chemical Society
ISSN: 00027863
DOI: 10.1021/ja058093d
Appears in Collections:Staff Publications

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