Microbiology (2000), 146, 1727–1734 Printed in Great Britain Serine and alanine racemase activities of VanT : a protein necessary for vancomycin resistance in Enterococcus gallinarum BM4174 Cesar A. Arias,† Jan Weisner, Jonathan M. Blackburn and Peter E. Reynolds Author for correspondence : Cesar A. Arias. Tel : 57 1 633 1512. Fax: 1 917 477 3388. e-mail : caa22cable.net.co Department of Biochemistry, University of Cambridge, Tennis Court Road, The Downing Site, Cambridge CB2 1QW, UK 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 SC 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 SC. No alanine or serine racemase activities were detected in the host strain E. coli TKL-10 grown at 30, 34 or 37 SC. 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 : -serine, vancomycin resistance, racemases, Enterococcus gallinarum INTRODUCTION Vancomycin is a glycopeptide antibiotic that inhibits synthesis of the bacterial cell wall by binding to the acyl- -alanyl--alanine (-Ala--Ala) moiety of peptido- glycan precursors (Reynolds, 1989).Vancomycin resist- ance in Enterococcus gallinarum results from the syn- thesis of precursors ending in -serine (-Ser) (Reynolds ................................................................................................................................................. † Present address : Centro de Investigaciones, Universidad El Bosque, Transversal 9A no. 133-25, Santafe  de Bogota  , Colombia. et al., 1994 ; Billot-Klein et al., 1994a) : the antibiotic has a lower affinity for UDP-MurNAc-pentapeptide[Ser] (the main peptidoglycan precursor synthesized) than for UDP-MurNAc-pentapeptide[Ala] (Billot Klein et al., 1994b). Resistance requires the presence of three pro- teins (Arias et al., 2000) : VanC-1 synthesizes the dipeptide -Ala--Ser (Park et al., 1997) that is in- corporated into peptidoglycan precursors ; VanXY C hydrolyses the dipeptide -Ala--Ala and removes - Ala from peptidoglycan precursors ending in -Ala, ensuring the removal of ‘ susceptible ’ precursors 0002-3904  2000 SGM 1727