Characterization and modelling of VanT: a novel, membrane-bound, serine racemase from vancomycin-resistant Enterococcus gallinarum BM4174

Sequence determination of a region downstream from the vanXY(c) gene in Ent erococcus gallinarum BM4174 revealed an open reading frame, designated vanT , that encodes a 698-amino-acid polypeptide with an amino-terminal domain c ontaining 10 predicted transmembrane segments. The protein contained a high ly conserved pyridoxal phosphate attachment site in the C-terminal domain, typical of alanine racemases. The protein was overexpressed in Escherichia coli, and serine racemase activity was detected in the membrane but not in the cytoplasmic fraction after centrifugation of sonicated cells, whereas a lanine racemase activity was located almost exclusively in the cytoplasm. W hen the protein was overexpressed as a polypeptide lacking the predicted tr ansmembrane domain, serine racemase activity was detected in the cytoplasm. The serine racemase activity was partially (64%) inhibited by D-cycloserin e, whereas host alanine racemase activity was almost totally inhibited (97% ). Serine racemase activity was also detected in membrane preparations of c onstitutively vancomycin-resistant E. gallinarum BM4174 but not in BM4175, in which insertional inactivation of the vanC-1 D-Ala:D-Ser ligase gene pro bably had a polar effect on expression of the vanXY(c) and vanT genes. Comp arative modelling of the deduced C-terminal domain was based on the alignme nt of VanT with the Alr alanine racemase from Bacillus stearothermophilus. The model revealed that almost all critical amino acids in the active site of Alr were conserved in VanT, indicating that the C-terminal domain of Van T is likely to adopt a three-dimensional structure similar to that of Alr a nd that the protein could exist as a dimer. These results indicate that the source of D-serine for peptidoglycan synthesis in vancomycin-resistant ent erococci expressing the VanC phenotype involves racemization of L- to D-ser ine by a membrane-bound serine racemase.