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

• Published in: Microbiology (Society for General Microbiology) Vol. 146; no. 7; pp. 1727 - 1734
• Main Authors: Arias, Cesar A, Weisner, Jan, Blackburn, Jonathan M, Reynolds, Peter E
• Format: Journal Article
• Language: English
• Published: Reading Soc General Microbiol 01.07.2000; Society for General Microbiology
• Subjects: Alanine Racemase - genetics; Alanine Racemase - metabolism; Amino Acid Sequence; Anti-Bacterial Agents - pharmacology; Antibacterial agents; Antibiotics. Antiinfectious agents. Antiparasitic agents; Bacillus - enzymology; Bacillus - genetics; Bacterial Proteins - genetics; Bacterial Proteins - isolation & purification; Bacterial Proteins - metabolism; Bacteriology; Biological and medical sciences; Blotting, Western; Enterococcus - drug effects; Enterococcus - enzymology; Enterococcus - genetics; Enterococcus gallinarum; Escherichia coli - genetics; Fundamental and applied biological sciences. Psychology; Medical sciences; Membrane Proteins - genetics; Membrane Proteins - isolation & purification; Membrane Proteins - metabolism; Metabolism. Enzymes; Microbiology; Molecular Sequence Data; Pharmacology. Drug treatments; Plasmids; Racemases and Epimerases - genetics; Racemases and Epimerases - isolation & purification; Racemases and Epimerases - metabolism; Sequence Alignment; serine racemase; Temperature; Transformation, Bacterial; Vancomycin - pharmacology; Vancomycin Resistance; VanT protein; Alanine racemase; Membrane protein; Peptides; Enzyme; Vancomycin; Structure function relationship; Mechanism; Resistance; Streptococcaceae; Antibiotic; Enterococcus gallinarum; Isomerases; Enzymatic activity; Glycopeptide; Bacteria; Micrococcales
• ISSN: 1350-0872; 1465-2080
• DOI: 10.1099/00221287-146-7-1727

Department of Biochemistry, University of Cambridge, Tennis Court Road, The Downing Site, Cambridge CB2 1QW, UK 1 Author for correspondence: Cesar A. Arias. Tel: +57 1 633 1512. Fax: +1 917 477 3388. e-mail: caa22{at}cable.net.co Vancomycin resistance in Enterococcus gallinarum 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 Escherichia coli TKL-10 that requires D-Ala for growth at 42 °C was used as host for transformation experiments using plasmids containing the full-length vanT from Ent. gallinarum or the alanine racemase gene ( alr ) of Bacillus stearothermophilus : both plasmids were able to complement E. coli TKL-10 at 42 °C. No alanine or serine racemase activities were detected in the host strain E. coli TKL-10 grown at 30, 34 or 37 °C. Serine and alanine racemase activities were found almost exclusively (96%) in the membrane fraction of E. coli TKL-10/pCA4( vanT ): the alanine racemase activity of VanT was 14% of the serine racemase activity in both E. coli TKL-10/pCA4( vanT ) and E. coli XL-1 Blue/pCA4( vanT ). Alanine racemase activity was present mainly (95%) in the cytoplasmic fraction of E. coli TKL-10/pJW40( alr ), with a trace (1·6%) of serine racemase activity. Additionally, DNA encoding the soluble domain of VanT was cloned and expressed in E. coli 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 E. coli 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. Keywords: D -serine, vancomycin resistance, racemases, Enterococcus gallinarum a Present address: Centro de Investigaciones, Universidad El Bosque, Transversal 9A no. 133-25, Santafé de Bogotá, Colombia.