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Volume 146, Issue 7

Serine and alanine racemase activities of VanT: a protein necessary for vancomycin resistance in BM4174 Free

Abstract

Vancomycin resistance in results from the production of UDP-MurNAc-pentapeptide[D-Ser]. VanT, a membrane-bound serine racemase, is one of three proteins essential for this resistance. To investigate the selectivity of racemization of L-Ser or L-Ala by VanT, a strain of TKL-10 that requires D-Ala for growth at 42 °C was used as host for transformation experiments using plasmids containing the full-length from or the alanine racemase gene () of : both plasmids were able to complement TKL-10 at 42 °C. No alanine or serine racemase activities were detected in the host strain TKL-10 grown at 30, 34 or 37 °C. Serine and alanine racemase activities were found almost exclusively (96%) in the membrane fraction of TKL-10/pCA4(): the alanine racemase activity of VanT was 14% of the serine racemase activity in both TKL-10/pCA4() and XL-1 Blue/pCA4(). Alanine racemase activity was present mainly (95%) in the cytoplasmic fraction of TKL-10/pJW40(), with a trace (16%) of serine racemase activity. Additionally, DNA encoding the soluble domain of VanT was cloned and expressed in M15 as a His-tagged polypeptide and purified: this polypeptide also exhibited both serine and alanine racemase activities; the latter was approximately 18% of the serine racemase activity, similar to that of the full-length, membrane-bound enzyme. N-terminal sequencing of the purified His-tagged polypeptide revealed a single amino acid sequence, indicating that the formation of heterodimers between subunits of His-tagged C-VanT and endogenous alanine racemases from was unlikely. The authors conclude that the membrane-bound serine racemase VanT also has alanine racemase activity but is able to racemize serine more efficiently than alanine, and that the cytoplasmic domain is responsible for the racemase activity.

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Keyword(s): d-serine , Enterococcus gallinarum , racemases and vancomycin resistance
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2000-07-01
2025-02-08
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Serine and alanine racemase activities of VanT: a protein necessary for vancomycin resistance in Enterococcus gallinarum BM4174
Microbiology 146, 1727 (2000); https://doi.org/10.1099/00221287-146-7-1727
/content/journal/micro/10.1099/00221287-146-7-1727
/content/journal/micro/10.1099/00221287-146-7-1727
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