Bio-Claisen condensation catalyzed by thiolase from Zoogloea ramigera. Active site cysteine residues

Masamune, Satoru; Palmer, Michelle; Gamboni, Remo; Thompson, Stuart A.; Davis, Jeffrey T.; Williams, Simon F.; Peoples, Oliver P.; Sinskey, Anthony J.; Walsh, Christopher T.

doi:10.1021/ja00187a053

Cited by 38 publications

(25 citation statements)

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“…As the reaction mechanism requires the abstraction of an a-proton to initiate nucleophilic attack, we do not expect all molecular pairs to be equally likely to form both isomers. Indeed, our inability to observe any alternative products in the synthesis of 3HV is consistent with a previous report that found no activity with a BktB homologue from Zoogloea ramigera (51% identity/ 68% similarity) in the thiolytic direction with the branched 2-methylacetoacetyl-CoA isomer of b-ketovaleryl-CoA 40 . The unexpected production of 2,3-DHBA is an exciting development because of the implication for other molecular structures that could be formed using substrates with sufficiently electronegative a-protons.…”

Section: Resultssupporting

confidence: 92%

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A platform pathway for production of 3-hydroxyacids provides a biosynthetic route to 3-hydroxy-γ-butyrolactone

et al. 2013

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The replacement of petroleum feedstocks with biomass to produce platform chemicals requires the development of appropriate conversion technologies. 3-Hydroxy-g-butyrolactone has been identified as one such chemical; however, there are no naturally occurring biosynthetic pathways for this molecule or its hydrolyzed form, 3,4-dihydroxybutyric acid. Here we design a novel pathway to produce various chiral 3-hydroxyacids, including 3,4-dihydroxybutyric acid, consisting of enzymes that condense two acyl-CoAs, stereospecifically reduce the resulting b-ketone and hydrolyze the CoA thioester to release the free acid. Acetyl-CoA serves as one substrate for the condensation reaction, whereas the second is produced intracellularly by a pathway enzyme that converts exogenously supplied organic acids. Feeding of butyrate, isobutyrate and glycolate results in the production of 3-hydroxyhexanoate, 3-hydroxy-4-methylvalerate and 3,4-dihydroxybutyric acid þ 3-hydroxy-gbutyrolactone, respectively, molecules with potential uses in applications from materials to medicines. We also unexpectedly observe the condensation reaction resulting in the production of the 2,3-dihydroxybutyric acid isomer, a potential value-added monomer.

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Section: Resultssupporting

confidence: 92%

“…2,3-DHBA was one possible isomeric product, identical in mass to 3,4-DHBA, that we hypothesized could be formed through an alternative Claisen condensation reaction 39 catalyzed by BktB 40 .…”

Section: Resultsmentioning

confidence: 94%

A platform pathway for production of 3-hydroxyacids provides a biosynthetic route to 3-hydroxy-γ-butyrolactone

et al. 2013

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“…Cys, Ala, and Ser have similar molecular volumes and intramolecular bonding interactions, which should minimize any perturbation of the three-dimensional structure of the TcmK protein. We thought that the C173S exchange might retain some activity due to the formation of O-ester bonds with the initial substrates and intermediates of the growing polyketide chain, as has been observed with a C89S mutant of biosynthetic thiolase enzymes from Zoogloea ramigera (21), whose catalytic mechanism resembles that proposed for the KSs. Instead, our results (Fig.…”

mentioning

confidence: 68%

Functional analysis of putative beta-ketoacyl:acyl carrier protein synthase and acyltransferase active site motifs in a type II polyketide synthase of Streptomyces glaucescens

Meurer

Hutchinson

1995

J Bacteriol

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The significance of potential active site motifs for acyltransferase and ␤-ketoacyl:acyl carrier protein synthase regions within the TcmK protein was investigated by determining the effects of mutations in the proposed active sites on the production of tetracenomycins F2 and C. In a Streptomyces glaucescens tcmGHI JKLMNO null mutant, plasmids carrying the S351A mutation produced high amounts of tetracenomycin F2 but plasmids carrying the C173A or C173S mutation or the H350L-S351A double mutation produced no detectable amount of any known intermediate. In a tcmK mutant, plasmids with the S351A mutation restored high production of tetracenomycin C and plasmids carrying the other mutations were able to complement the chromosomal defect to some extent. None of the mutations affected the amount of TcmK produced.

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“…Comparison of several KS domains indicates that most KS domains contain a conserved cysteine in the motif, which is the presumed active site, as seen in Fig. In addition to these examples, it has been observed that a Cys-89 to Ser mutant of biosynthetic thiolase enzymes from Zoogloea ramigera, whose catalytic mechanism resembles that proposed for the KSs, retained its activity [36]. Cys and Ser have similar molecular volumes and intermolecular bonding interactions, and therefore, this change minimizes any perturbation of the three-dimensional structure of TaK in comparison to other KS domains.…”

Section: Discussionmentioning

confidence: 83%

An unusual β-ketoacyl:acyl carrier protein synthase and acyltransferase motifs in TaK, a putative protein required for biosynthesis of the antibiotic TA in Myxococcus xanthus

Paitan¹

2001

FEMS Microbiology Letters

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The antibiotic TA of Myxococcus xanthus is produced by a type‐I polyketide synthase mechanism. Previous studies have indicated that TA genes are clustered within a 36‐kb region. The chemical structure of TA indicates the need for several post‐modification steps, which are introduced to form the final bioactive molecule. These include three C‐methylations, an O‐methylation and a specific hydroxylation. In this study, we describe the genetic analysis of taK, encoding a specific polyketide β‐ketoacyl:acyl carrier protein synthase, which contains an unusual β‐ketoacyl synthase and acyltransferase motifs and is likely to be involved in antibiotic TA post‐modification. Functional analysis of this β‐ketoacyl:acyl carrier protein synthase by specific gene disruption suggests that it is essential for the production of an active TA molecule.

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Bio-Claisen condensation catalyzed by thiolase from Zoogloea ramigera. Active site cysteine residues

Cited by 38 publications

References 0 publications

A platform pathway for production of 3-hydroxyacids provides a biosynthetic route to 3-hydroxy-γ-butyrolactone

A platform pathway for production of 3-hydroxyacids provides a biosynthetic route to 3-hydroxy-γ-butyrolactone

Functional analysis of putative beta-ketoacyl:acyl carrier protein synthase and acyltransferase active site motifs in a type II polyketide synthase of Streptomyces glaucescens

An unusual β-ketoacyl:acyl carrier protein synthase and acyltransferase motifs in TaK, a putative protein required for biosynthesis of the antibiotic TA in Myxococcus xanthus

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