Mutational analysis of active-site residues of the enterococcal D-Ala-D-Ala dipeptidase VanX and comparison with Escherichia coli D-Ala-D-Ala ligase and D-Ala-D-Ala carboxypeptidase VanY

Background: Vancomycin-resistant enterococci are pathogenic bacteria that a ttenuate antibiotic sensitivity by producing peptidoglycan precursors that terminate in D-Ala-D-lactate rather than D-Ala-D-Ala. A key enzyme in effec ting antibiotic resistance is the metallodipeptidase VanX, which reduces th e cellular pool of the D-Ala-D-Ala dipeptide. Results: We constructed eleven mutants, using the recently determined VanX structure as a basis, to investigate residue function. Mutating Asp142 or S er114 showed a large effect principally on K-M, consistent with roles in re cognition of the D-Ala-D-Ala termini. The drastic reduction or absence of a ctivity in the Arg71 mutants correlates with a role in the stabilization of an anionic tetrahedral transition state. Three residues of the Escherichia coli D-Ala-D-Ala ligase (Ddl), Glu15, Ser281 and Arg255, are similarly con served and have equivalent functions with respect to VanX, consistent with a convergent evolution of active sites to bind D-Ala-D-Ala and tower energy barriers for formation of the tetrahedral intermediate and transition stat es. In the N-acyl-D-Ala-D-Ala carboxypeptidase VanY, all active-site residu es are conserved (except for the two responsible for recognition of the dip eptide amino terminus). Conclusions: The mutagenesis results support structure-based functional pre dictions and explain why the VanX dipeptidase and Ddl ligase show narrow sp ecificity for the D,D-dipeptide substrate, The results reveal that VanX and Ddl, two enzymes that use the same substrate but proceed in opposite direc tions driven by distinct cofactors (zinc versus AIP), evolved similar archi tectural solutions to substrate recognition and catalysis acceleration. Van Y sequence analysis predicts an active site and mechanism of reaction simil ar to VanX.