Stereospecificity of the Dehydratase Domain of the Erythromycin Polyketide Synthase

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• 作者：Chiara R. Valenzano ; Young-Ok You ; Ashish Garg ; Adrian Keatinge-Clay ; Chaitan Khosla ; David E. Cane
• 刊名：Journal of the American Chemical Society
• 出版年：2010
• 出版时间：October 27, 2010
• 年：2010
• 卷：132
• 期：42
• 页码：14697-14699
• 全文大小：191K
• 年卷期：v.132,no.42(October 27, 2010)
• ISSN：1520-5126

文摘

The dehydratase (DH) domain of module 4 of the 6-deoxyerythronolide B synthase (DEBS) has been shown to catalyze an exclusive syn elimination/syn addition of water. Incubation of recombinant DH4 with chemoenzymatically prepared anti-(2R,3R)-2-methyl-3-hydroxypentanoyl-ACP (2a-ACP) gave the dehydration product 3-ACP. Similarly, incubation of DH4 with synthetic 3-ACP resulted in the reverse enzyme-catalyzed hydration reaction, giving an 3:1 equilbrium mixture of 2a-ACP and 3-ACP. Incubation of a mixture of propionyl-SNAC (4), methylmalonyl-CoA, and NADPH with the DEBS β-ketoacyl synthase-acyl transferase [KS6][AT6] didomain, DEBS ACP6, and the ketoreductase domain from tylactone synthase module 1 (TYLS KR1) generated in situ anti-2a-ACP that underwent DH4-catalyzed syn dehydration to give 3-ACP. DH4 did not dehydrate syn-(2S,3R)-2b-ACP, syn-(2R,3S)-2c-ACP, or anti-(2S,3S)-2d-ACP generated in situ by DEBS KR1, DEBS KR6, or the rifamycin synthase KR7 (RIFS KR7), respectively. Similarly, incubation of a mixture of (2S,3R)-2-methyl-3-hydroxypentanoyl-N-acetylcysteamine thioester (2b-SNAC), methylmalonyl-CoA, and NADPH with DEBS [KS6][AT6], DEBS ACP6, and TYLS KR1 gave anti-(2R,3R)-6-ACP that underwent syn dehydration catalyzed by DEBS DH4 to give (4R,5R)-(E)-2,4-dimethyl-5-hydroxy-hept-2-enoyl-ACP (7-ACP). The structure and stereochemistry of 7 were established by GC-MS and LC-MS comparison of the derived methyl ester 7-Me to a synthetic sample of 7-Me.