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PH-Rate profiles establish that polyketide synthase dehydratase domains utilize a single-base mechanism.

ABSTRACT: FosDH1 from module 1 of the fostriecin polyketide synthase (PKS) catalyzes the dehydration of a 3-hydroxybutyryl-SACP to the (E)-3-butenoyl-SACP. The steady-state kinetic parameters, kcat and kcat/Km, were determined over the pH range 3.0 to 9.2 for the FosDH1-catalyzed dehydration of the N-acetycsteamine thioester, 3-hydroxybutyryl-SNAC (3), to (E)-3-butenoyl-SNAC (4). The pH rate profiles for both log(kcat) and log(kcat/Km) each corresponded to a single pH-dependent ionization to give an active site general base, with a calculated pKa 6.1 ± 0.2 for kcat and pKa 5.7 ± 0.1 for kcat/Km. These results are inconsistent with the commonly suggested "two-base" (base-acid) mechanism for the dehydratases of PKS and fatty acid biosynthesis and support a simple one-base mechanism in which the universally conserved active site His residue acts as the base to deprotonate C-2 of the substrate, then redonates the proton to the C-3 hydroxyl group to promote C-O bond-cleavage and elimination of water. The carboxylate of the paired Asp or Glu residue is thought to bind and orient the hydroxyl group of the substrate in the stereoelectonically favored conformation.

SUBMITTER: Xie X

PROVIDER: S-EPMC6295148 | biostudies-literature | 2018 Dec

REPOSITORIES: biostudies-literature

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pH-Rate profiles establish that polyketide synthase dehydratase domains utilize a single-base mechanism.

Xie Xinqiang X Cane David E DE

Organic & biomolecular chemistry 20181201 47

FosDH1 from module 1 of the fostriecin polyketide synthase (PKS) catalyzes the dehydration of a 3-hydroxybutyryl-SACP to the (E)-3-butenoyl-SACP. The steady-state kinetic parameters, kcat and kcat/Km, were determined over the pH range 3.0 to 9.2 for the FosDH1-catalyzed dehydration of the N-acetycsteamine thioester, 3-hydroxybutyryl-SNAC (3), to (E)-3-butenoyl-SNAC (4). The pH rate profiles for both log(kcat) and log(kcat/Km) each corresponded to a single pH-dependent ionization to give an activ ...[more]

PMID: 30457629

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Project description:RifDH10, the dehydratase domain from the terminal module of the rifamycin polyketide synthase, catalyzes the stereospecific syn dehydration of the model substrate (2S,3S)-2-methyl-3-hydroxypentanoyl-RifACP10, resulting in the exclusive formation of (E)-2-methyl-2-pentenoyl-RifACP10. RifDH10 does not dehydrate any of the other three diastereomeric, RifACP10-bound, diketide thioester substrates. On the other hand, when EryACP6, from the sixth module of the erythromycin polyketide synthase, is substituted for RifACP10, RifDH10 stereospecifically dehydrates only (2R,3R)-2-methyl-3-hydroxypentanoyl-EryACP6 to give exclusively (E)-2-methyl-2-pentenoyl-EryACP6, with no detectable dehydration of any of the other three diastereomeric, EryACP6-bound, diketides. An identical alteration in substrate diastereospecificity was observed for the corresponding N-acetylcysteamine or pantetheine thioester analogues, regardless of acyl chain length or substitution pattern. Incubation of (2RS)-2-methyl-3-ketopentanoyl-RifACP10 with the didomain reductase-dehydratase RifKR10-RifDH10 yielded (E)-2-methyl-2-pentenoyl-RifACP10, the expected product of syn dehydration of (2S,3S)-2-methyl-3-hydroxypentanoyl-RifACP10, while incubation with the corresponding EryACP6-bound substrate, (2RS)-2-methyl-3-ketopentanoyl-EryACP6, gave only the reduction product (2S,3S)-2-methyl-3-hydroxypentanoyl-EryACP6 with no detectable dehydration. These results establish the intrinsic syn dehydration stereochemistry and substrate diastereoselectivity of RifDH10 and highlight the critical role of the natural RifACP10 domain in chaperoning the proper recognition and processing of the natural ACP-bound undecaketide substrate. The 1.82 Å resolution structure of RifDH10 reveals the atomic-resolution details of the active site and allows modeling of the syn dehydration of the (2S,3S)-2-methyl-3-hydroxyacyl-RifACP10 substrate. These results suggest that generation of the characteristic cis double bond of the rifamycins occurs after formation of the full-length RifACP10-bound acyclic trans-unsaturated undecaketide intermediate, most likely during the subsequent macrolactamization catalyzed by the amide synthase RifF.

Dataset Information

PH-Rate profiles establish that polyketide synthase dehydratase domains utilize a single-base mechanism.

Publications

pH-Rate profiles establish that polyketide synthase dehydratase domains utilize a single-base mechanism.

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