ABSTRACT
D-Alanine-D-alanine ligase is the second enzyme in the
D-Ala branch of bacterial cell wall peptidoglycan assem-
bly, and recognized as an attractive antimicrobial target.
In this work, the D-Ala-D-Ala ligase of Helicobacter
pylori strain SS1 (HpDdl) was kinetically and structur-
ally characterized. The determined apparent Km of ATP
(0.87 lM), the Km1 (1.89 mM) and Km2 of D-Ala (627
mM), and the kcat (115 min21) at pH 8.0 indicated its
relatively weak binding affinity and poor catalytic activ-
ity against the substrate D-Ala in vitro. However, by
complementary assay of expressing HpDdl in Escherichia
coli Dddl mutant, HpDdl was confirmed to be capable
of D-Ala-D-Ala ligating in vivo. Through sequence align-
ment with other members of the D-Ala-D-X ligase super-
family, HpDdl keeps two conservatively substituted resi-
dues (Ile16 and Leu241) and two nonconserved residues
(Leu308 and Tyr311) broadly located in the active region
of the enzyme. Kinetic analyses against the correspond-
ing HpDdl mutants (I16V, L241Y, L241F, L308T, and
Y311S) suggested that these residues, especially Leu308
and Tyr311, might partly contribute to the unique cata-
lytic properties of the enzyme. This was fairly proved by
the crystal structure of HpDdl, which revealed that there
is a 310-helix (including residues from Gly306 to
Leu312) near the D-Ala binding region in the C-termi-
nal domain, where HpDdl has two sequence deletions
compared with other homologs. Such 310-helix may par-
ticipate in D-Ala binding and conformational change of
the enzyme. Our present work hopefully provides useful
information for understanding the D-Ala-D-Ala ligase of
Helicobacter pylori.