PDBsum entry 2pvp

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Ligase PDB id
Protein chains
329 a.a.
Waters ×95

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Key reference
Title Enzymatic characterization and crystal structure analysis of the d-Alanine-D-Alanine ligase from helicobacter pylori.
Authors D.Wu, L.Zhang, Y.Kong, J.Du, S.Chen, J.Chen, J.Ding, H.Jiang, X.Shen.
Ref. Proteins, 2008, 72, 1148-1160. [DOI no: 10.1002/prot.22009]
PubMed id 18320587
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 muM), 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 Deltaddl 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. Proteins 2008. (c) 2008 Wiley-Liss, Inc.
Figure 5.
Figure 5. Overall structure of HpDdl in stereoview. (A) Three domains of HpDdl monomer are colored in green, yellow and red (from N to C-terminus), respectively. The 3[10]-helix is colored in cyan and the location of putative active site is labeled by the arrow. (B) The 3[10]-helix (sticks) embedded in 2F[o] - F[c] type electron density map contoured at 1.0 (blue). Carbon, oxygen and nitrogen are colored cyan, red and blue respectively. Residues in the 3[10]-helix are labeled. (C) Structure comparison after superposition with the C-terminal domains of HpDdl (cyan), EcDdlB (green, PDB code 2DLN) and ThcDdl (magenta, PDB code 2FB9). The -loop and 3[10]-helix regions of HpDdl are indicated.
Figure 6.
Figure 6. Active site of HpDdl in stereoview. A: Superposition of some 3[10]-helix and mutated residues in HpDdl (cyan) and their counterparts (green), ADP and the phosphorylated phosphinate (magenta) in EcDdlB.[20] Ligands and residues are labeled. The secondary structures near the residues are also shown in cartoon model. B: The hydrophobic environment (yellow) around Leu308 in HpDdl. Phosphinate (magenta) and residues are labeled. The secondary structure of the 3[10]-helix is also shown in cartoon model.
The above figures are reprinted by permission from John Wiley & Sons, Inc.: Proteins (2008, 72, 1148-1160) copyright 2008.
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