EVOLUTION OF STRUCTURE AND SUBSTRATE-SPECIFICITY IN D-ALANINE-D-ALANINE LIGASES AND RELATED ENZYMES

The D-alanine:D-alanine-ligase-related enzymes can have three preferen tial substrate specificities. Usually, these enzymes synthesize D-alan yl-D-alanine. In vancomycin-resistant Gram-positive bacteria, structur ally related enzymes synthesize D-alanyl-D-lactate or D-alanyl-D-serin e. The sequence of internal fragments of eight structural D-alanine:D- alanine ligase genes from enterococci has been determined. Alignment o f the deduced amino acid sequences with those of other related enzymes from Gram-negative and Gram-positive bacteria revealed the presence o f four distinct sequence patterns in the putative substrate-binding si tes, each correlating with specificity to a particular substrate (D-al anine:D-lactate li,oases exhibited two patterns), Phylogenetic analysi s showed different clusters. The enterococcal subtree was largely supe rimposable on that derived from 16S rRNA sequences. In lactic acid bac teria, structural divergence due to differences in substrate specifici ty was observed. Glycopeptide resistance proteins VanA and VanB, the V anC-type ligases, and DdlA and DdlB from enteric bacteria and Haemophi lus influenzae constituted separate clusters.