Wu Dalei, Zhang Liang, Kong Yunhua, Du Jiamu, Chen Shuai, Chen Jing, Ding Jianping, Jiang Hualiang, Shen Xu
Drug Discovery and Design Center, State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China.
Proteins. 2008 Sep;72(4):1148-60. doi: 10.1002/prot.22009.
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.
D-丙氨酸-D-丙氨酸连接酶是细菌细胞壁肽聚糖组装的D-丙氨酸分支中的第二种酶，被认为是一个有吸引力的抗菌靶点。在这项工作中，对幽门螺杆菌菌株SS1的D-丙氨酸-D-丙氨酸连接酶（HpDdl）进行了动力学和结构表征。在pH 8.0条件下测定的ATP的表观K(m)（0.87微摩尔）、D-丙氨酸的K(m1)（1.89毫摩尔）和K(m2)（627毫摩尔）以及k(cat)（115分钟(-1)）表明其在体外对底物D-丙氨酸的结合亲和力相对较弱且催化活性较差。然而，通过在大肠杆菌Delta ddl突变体中表达HpDdl的互补试验，证实HpDdl在体内能够进行D-丙氨酸-D-丙氨酸连接。通过与D-丙氨酸-D-X连接酶超家族的其他成员进行序列比对，HpDdl保留了两个保守取代的残基（Ile16和Leu241）以及两个非保守残基（Leu308和Tyr311），它们广泛位于酶的活性区域。对相应的HpDdl突变体（I16V、L241Y、L241F、L308T和Y311S）的动力学分析表明，这些残基，尤其是Leu308和Tyr311，可能部分促成了该酶独特的催化特性。HpDdl的晶体结构充分证明了这一点，该结构显示在C端结构域的D-丙氨酸结合区域附近存在一个3(10)-螺旋（包括从Gly306到Leu312的残基），与其他同源物相比，HpDdl在此处有两个序列缺失。这样的3(10)-螺旋可能参与D-丙氨酸的结合以及酶的构象变化。我们目前的工作有望为理解幽门螺杆菌的D-丙氨酸-D-丙氨酸连接酶提供有用信息。
