J Mol Microbiol Biotechnol 2000 Jul;2(3):321-30

Modeling of Enterococcus faecalis D-alanine:D-alanine ligase: structure-based
study of the active site in the wild-type enzyme and in glycopeptide-dependent
mutants.

Prevost M, Van Belle D, Tulkens PM, Courvalin P, Van Bambeke F

Ingenierie Biomoleculaire, Universite Libre de Bruxelles, Brussels, Belgium.
mprevost@ulb.ac.be

A model for the 3-D structure of Enterococcus faecalis D-Ala:D-Ala ligase was
produced using the X-ray structure of the Escherichia coli enzyme complexed with
ADP and the methylphosphinophosphate inhibitor as a template. The model passed
critical validation criteria with an accuracy similar to that of the template
crystallographic structure and showed that ADP and methylphosphinophosphate were
positioned in a large empty pocket at the interface between the central and the
C-terminal domains, as in E. coli. It evidenced the residues important for
substrate binding and catalytic activity in the active site and demonstrated a
large body of conserved interactions between the active sites of the E. faecalis
and the E. coli D-Ala:D-Ala ligase, the major differences residing in the
balance between the hydrophobic and aromatic environment of the adenine. The
model also successfully explained the inactivity of four spontaneous mutants
(D295 --> V, which impairs interactions with Mg2+ and R293, which are both
essential for binding and catalytic activity; S319 --> I, which perturbs
recognition of D-Ala2; DAK251-253 --> E, in which the backbone conformation in
the vicinity of the deletion remains unaltered but phosphate transfer from ATP
is perturbed because of lack of K253; T316 --> I, which causes the loss of a
hydrogen bond affecting the positioning of S319 and therefore the binding of
D-Ala2). Since D-Ala:D-Ala ligase is an essential enzyme for bacteria, this
approach, combining molecular modeling and molecular biology, may help in the
design of specific ligands which could inhibit the enzyme and serve as novel
antibiotics.

PMID: 10937441, UI: 20391161