Enzymatic characterization and crystal structure analysis of the D‐alanine‐D‐alanine ligase from Helicobacter pylori

Abstract

D-Alanine-D-alanine ligase is the second enzyme in the D-Ala branch of bacterial cell wall peptidoglycan assembly, 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 structurally characterized. The determined apparent K(m) of ATP (0.87 microM), the K(m1) (1.89 mM) and K(m2) of D-Ala (627 mM), and the k(cat) (115 min(-1)) at pH 8.0 indicated its relatively weak binding affinity and poor catalytic activity against the substrate D-Ala in vitro. However, by complementary assay of expressing HpDdl in Escherichia coli Delta ddl mutant, HpDdl was confirmed to be capable of D-Ala-D-Ala ligating in vivo. Through sequence alignment with other members of the D-Ala-D-X ligase superfamily, HpDdl keeps two conservatively substituted residues (Ile16 and Leu241) and two nonconserved residues (Leu308 and Tyr311) broadly located in the active region of the enzyme. Kinetic analyses against the corresponding HpDdl mutants (I16V, L241Y, L241F, L308T, and Y311S) suggested that these residues, especially Leu308 and Tyr311, might partly contribute to the unique catalytic properties of the enzyme. This was fairly proved by the crystal structure of HpDdl, which revealed that there is a 3(10)-helix (including residues from Gly306 to Leu312) near the D-Ala binding region in the C-terminal domain, where HpDdl has two sequence deletions compared with other homologs. Such 3(10)-helix may participate 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.

Structure of the Mycobacterium tuberculosis d -Alanine: d -Alanine Ligase, a Target of the Antituberculosis Drug d -Cycloserine

“…This value is similar to the IC 50 for E. coli DdlB at 0.32 mM (21). The value of the second binding event for DCS is similar to that of the M. tuberculosis IC 50 and published IC 50 s for the E. coli Ddl (21,45).…”

“…Structure-based drug design using DCS as a scaffold could aid in developing Ddl inhibitors with greater specificity and attenuated severity of side effects. Unfortunately, no atomic level structural information regarding M. tuberculosis Ddl has been reported, although Ddl structures from other organisms are available (13,23,25,45).…”

d-Alanine:d-alanine ligase as a new target for the flavonoids quercetin and apigenin

“…In our recent work, we discovered that these two flavonoids could inhibit the FabZ enzyme from H. pylori [11], a human pathogen associated with many diseases involving gastroduodenal tissue, e.g., gastritis, peptic ulceration and gastric cancer [12]. d-Alanine:d-alanine ligase (Ddl) is an essential enzyme that catalyses the ligation of d-Ala-d-Ala in the assembly of peptidoglycan precursors [13] and has been considered as an important antimicrobial drug target for years [14]. To date, nearly all the reported Ddl inhibitors are Ala analogues (e.g.…”

Crystallization and preliminary X-ray analysis of aD-alanyl-D-alanine ligase (EcDdlB) fromEscherichia coli

“…Therefore, the study of these enzymes is of prime interest in the search for new antimicrobials, particularly by rational drug-design methods. In order to elucidate the structural and chemical basis of enzyme activity, the crystal structures of Ddl from Escherichia coli (Fan et al, 1994), Staphylococcus aureus (Liu et al, 2006), Thermus caldophilus (Lee et al, 2006), Helicobacter pylori (Wu et al, 2008) and T. thermophilus HB8 (Kitamura et al, 2009) have been reported. Additionally, the crystal structures of d-alanyld-lactate ligases from Enterococcus faecium (Roper et al, 2000) and Leuconostoc mesenteroides (Kuzin et al, 2000) have been reported.…”