Structure ofD-alanine-D-alanine ligase fromThermus thermophilusHB8: cumulative conformational change and enzyme–ligand interactions

Abstract

d-Alanine-d-alanine ligase (Ddl) is one of the key enzymes in peptidoglycan biosynthesis and is an important target for drug discovery. The enzyme catalyzes the condensation of two d-Ala molecules using ATP to produce d-Ala-d-Ala, which is the terminal peptide of a peptidoglycan monomer. The structures of five forms of the enzyme from Thermus thermophilus HB8 (TtDdl) were determined: unliganded TtDdl (2.3 Å resolution), TtDdl-adenylyl imidodiphosphate (2.6 Å ), TtDdl-ADP (2.2 Å ), TtDdl-ADP-d-Ala (1.9 Å ) and TtDdl-ATP-d-Ala-d-Ala (2.3 Å ). The central domain rotates as a rigid body towards the active site in a cumulative manner in concert with the local conformational change of three flexible loops depending upon substrate or product binding, resulting in an overall structural change from the open to the closed form through semi-open and semi-closed forms. Reaction-intermediate models were simulated using TtDdlcomplex structures and other Ddl structures previously determined by X-ray methods. The catalytic process accompanied by the cumulative conformational change has been elucidated based on the intermediate models in order to provide new insights regarding the details of the catalytic mechanism.

Structural and enzymatic characterization of BacD, an L‐amino acid dipeptide ligase from Bacillus subtilis

“…In contrast, Mg 2+ ‐2 shows an asymmetric coordination geometry with the carboxy groups of Glu109 and Glu324, the main‐chain carbonyl group of Leu182, and a water molecule. The binding mode of the enzyme with the phosphate groups of ADP, the two magnesium ions, and the phosphate group of the P‐analog is quite similar with those of Ddls and VanA as previously reported 14, 21, 22, 26…”

“…As in the case of DdlB‐related enzymes, the N‐terminal amino acid ( L ‐alanine) first binds to the ATP‐bound state of the enzyme. The previous structural study of Ddl from Thermus thermophilus HB8 in various substrate bound states (PDB IDs: 2YZG, 2YZN, 2ZDG, 2ZDH, and 2ZDQ) has suggested that the binding of the first D ‐alanine should be preceded by the binding of ATP because the loops forming the active site cannot take the conformation suitable for the amino‐acid binding without the ATP binding 26. The bound L ‐alanine is oriented for the phosphate transfer reaction with the aid of Glu273, His301, Glu311, and Gly331, and the carboxy oxygen atom nucleophilically attacks the phosphorous atom in the γ phosphate group [Figs.…”

Molecular Basis of Vancomycin Dependence in VanA-Type Staphylococcus aureus VRSA-9

“…This difference could explain the lower affinity for ATP of VRSA-9 Ddl. In a recent work (17), the crystal structure of the Ddl from Thermus thermophilus HB8 identified four conformational states (open, semiopen, semiclosed, and closed), and the reaction catalyzed by the Ddl is considered to proceed through these states. Upon binding of the substrate, the central domain rotates as a rigid body toward the C-terminal domain.…”

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.…”