Stereoselectivity of Isolated Dehydratase Domains of the Borrelidin Polyketide Synthase: Implications for <i>cis</i> Double Bond Formation

Vergnolle, Olivia; Hahn, Frank; Baerga‐Ortiz, Abel; Leadlay, Peter F.; Andexer, Jennifer N.

doi:10.1002/cbic.201100011

Cited by 42 publications

(49 citation statements)

References 35 publications

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“…Various nitriles are distributed in nature (e.g., indole-3-acetonitrile, ricinine, toyocamycin, and borrelidin), the nitrile group of which would be synthesized from the corresponding aldoxime (47)(48)(49). Comparison of the reaction mechanisms of borrelidin (nitrile)-synthesizing enzyme and aldoxime dehydratases (OxdA and OxdRE) could lead to the efficient production of useful nitriles.…”

Section: Discussionmentioning

confidence: 99%

Crystal structure of aldoxime dehydratase and its catalytic mechanism involved in carbon-nitrogen triple-bond synthesis

Nomura

Hashimoto

Ohta

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

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Aldoxime dehydratase (OxdA), which is a unique heme protein, catalyzes the dehydration of an aldoxime to a nitrile even in the presence of water in the reaction mixture. Unlike the utilization of H 2 O 2 or O 2 as a mediator of catalysis by other heme-containing enzymes (e.g., P450), OxdA is notable for the direct binding of a substrate to the heme iron. Here, we determined the crystal structure of OxdA. We then constructed OxdA mutants in which each of the polar amino acids lying within ∼6 Å of the iron atom of the heme was converted to alanine. Among the purified mutant OxdAs, S219A had completely lost and R178A exhibited a reduction in the activity. Together with this finding, the crystal structural analysis of OxdA and spectroscopic and electrostatic potential analyses of the wildtype and mutant OxdAs suggest that S219 plays a key role in the catalysis, forming a hydrogen bond with the substrate. Based on the spatial arrangement of the OxdA active site and the results of a series of mutagenesis experiments, we propose the detailed catalytic mechanism of general aldoxime dehydratases: (i) S219 stabilizes the hydroxy group of the substrate to increase its basicity; (ii) H320 acts as an acid-base catalyst; and (iii) R178 stabilizes the heme, and would donate a proton to and accept one from H320.hemoprotein | reaction mechanism | resonance Raman | distal histidine

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Section: Discussionmentioning

confidence: 99%

Crystal structure of aldoxime dehydratase and its catalytic mechanism involved in carbon-nitrogen triple-bond synthesis

Nomura

Hashimoto

Ohta

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

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“…Double bonds of the cis configuration are less common but nonetheless occur in a number of polyketides and arise through at least two mechanisms. In some cases, such as epothilone (Tang et al, 2003), borrelidin (Vergnolle et al, 2011) and hypothemycin (Reeves et al, 2008) biosynthesis, cis double bonds are formed by an enzyme-catalyzed isomerization of trans double bonds. In other pathways, such as the phoslactomycin (Alhamadsheh et al, 2007), rifamycin (Schupp et al, 1998), and fostriecin (Kong et al, 2013) synthases, it has been postulated that cis double bonds arise from the DH-catalyzed dehydration of an L-3-hydroxyacyl intermediate, which is formed by an A-type KR domain (Reid et al, 2003).…”

Section: Introductionmentioning

confidence: 99%

Structural and Stereochemical Analysis of a Modular Polyketide Synthase Ketoreductase Domain Required for the Generation of a cis-Alkene

Bonnett

Whicher

Kancharla

et al. 2013

Chemistry & Biology

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SUMMARY The formation of an activated cis-3-cyclohexylpropenoic acid by Plm1, the first extension module of the phoslactomycin PKS, is proposed to occur through an L-3-hydroxyacyl-intermediate as a result of ketoreduction by an A-type ketoreductase (KR). Here, we demonstrate that the KR domain of Plm1 (PlmKR1) catalyzes the formation of an L-3-hydroxyacyl product. The crystal structure of PlmKR1 revealed a well ordered active site with a nearby Trp residue characteristic of A-type KRs. Structural comparison of PlmKR1 with B-type KRs that produce D-3-hydroxyacyl intermediates revealed significant differences. The active site of cofactor-bound A-type KRs is in a catalysis-ready state, whereas cofactor-bound B-type KRs are in a pre-catalytic state. Furthermore, the closed lid loop in substrate-bound A-type KRs restricts active site access from all but one direction, which is proposed to control the stereochemistry of ketoreduction.

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“…14, 15 As a further complication, the formation of some c is double bonds can involve post-PKS transformations such as dehydration 7 or double-bond isomerization. 30 …”

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confidence: 99%

Structure and Stereospecificity of the Dehydratase Domain from the Terminal Module of the Rifamycin Polyketide Synthase

et al. 2013

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RifDH10, the dehydratase domain from the terminal module of the rifamycin polyketide synthase, catalyzed the stereospecific syn dehydration of the model substrate (2S,3S)-2-methyl-3-hydroxypentanoyl-RifACP10, resulting in exclusive formation of (E)-2-methyl-2-pentenoyl-RifACP10. RifDH10 did 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, was substituted for RifACP10, RifDH10 stereospecifically dehydrated 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 revealed the atomic resolution details of the active site and allowed 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.

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Stereoselectivity of Isolated Dehydratase Domains of the Borrelidin Polyketide Synthase: Implications for cis Double Bond Formation

Cited by 42 publications

References 35 publications

Crystal structure of aldoxime dehydratase and its catalytic mechanism involved in carbon-nitrogen triple-bond synthesis

Crystal structure of aldoxime dehydratase and its catalytic mechanism involved in carbon-nitrogen triple-bond synthesis

Structural and Stereochemical Analysis of a Modular Polyketide Synthase Ketoreductase Domain Required for the Generation of a cis-Alkene

Structure and Stereospecificity of the Dehydratase Domain from the Terminal Module of the Rifamycin Polyketide Synthase

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