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Issue 11, 2012
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Characterization of DcsC, a PLP-independent racemase involved in the biosynthesis of d-cycloserine

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Abstract

The biosynthetic gene cluster responsible for the generation of the antibiotic D-cycloserine (DCS) has recently been disclosed. One of the putative enzymes described was DcsC, which showed a high degree of homology to diaminopimelate epimerase (DapF). Based on this homology, the activity of DcsC was presumed to be the racemization of O-ureido-L-serine, a proposed intermediate in DCS biosynthesis. Here we describe the cloning, overexpression and characterization of this enzyme. Using synthetic standards we show that DcsC is a racemase that operates on both O-ureido-L- and D-serine, and that it employs a two-base mechanism, with a thiolate–thiol pair in the active site. The activity of this enzyme was shown to be optimal at pH ∼ 7.8, with a similar kcat/KM ratio in both the LD direction and DL direction. Activity was abolished with thiol-inactivating reagents such as iodoacetamide and Hg2+ ions. Further evidence for a thiolate in the active site was obtained through the use of an epoxide-containing substrate analogue (6), which became covalently attached to the enzyme.

Graphical abstract: Characterization of DcsC, a PLP-independent racemase involved in the biosynthesis of d-cycloserine

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Supplementary files

Article information


Submitted
04 Nov 2011
Accepted
05 Jan 2012
First published
06 Feb 2012

Org. Biomol. Chem., 2012,10, 2248-2254
Article type
Paper

Characterization of DcsC, a PLP-independent racemase involved in the biosynthesis of D-cycloserine

D. Dietrich, M. J. van Belkum and J. C. Vederas, Org. Biomol. Chem., 2012, 10, 2248
DOI: 10.1039/C2OB06864H

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