Stereospecificity of the dehydratase domain of the erythromycin polyketide synthase.

Abstract

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.

• Authors: Chiara R Valenzano, Young-Ok You, Ashish Garg, Adrian Keatinge-Clay, Chaitan Khosla, David E Cane
• Journal: Journal of the American Chemical Society (J Am Chem Soc) Vol. 132 Issue 42 Pg. 14697-9 (Oct 27 2010) ISSN: 1520-5126 [Electronic] United States
• PMID: 20925342 (Publication Type: Journal Article, Research Support, N.I.H., Extramural, Research Support, Non-U.S. Gov't)

Chemical References

• Acyl Carrier Protein
• Recombinant Proteins
• NADP
• Polyketide Synthases

Topics

• Acyl Carrier Protein (metabolism)
• Amino Acid Sequence
• Biocatalysis
• Chromatography, Gas
• Chromatography, Liquid
• Mass Spectrometry (methods)
• Models, Molecular
• Mutagenesis, Site-Directed
• NADP (metabolism)
• Polyketide Synthases (chemistry, genetics, metabolism)
• Recombinant Proteins (chemistry, genetics, metabolism)
• Stereoisomerism