The catalytic intermediate stabilized by a "Down" active site loop for diaminopimelate decarboxylase from Helicobacter pylori -: Enzymatic characterization with crystal structure analysis
The catalytic intermediate stabilized by a "Down" active site loop for diaminopimelate decarboxylase from Helicobacter pylori -: Enzymatic characterization with crystal structure analysis
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DOI：
10.1074/jbc.m801823200
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发表时间：
2008-07-25
影响因子：
4.8
通讯作者：
Shen, Xu
中科院分区：
文献类型：
--
作者：
Hu, Tiancen;Wu, Dalei;Shen, Xu
内消旋二氨基庚二酸脱羧酶（DAPDC，EC 4.1.1.20）催化细菌中 L-赖氨酸生物合成的最后一步，被视为抗生素发现的靶标。在这里，我们报道了来自幽门螺杆菌 (HpDAPDC) 的 DAPDC 的 2.3 埃晶体结构。该结构中，产物L-赖氨酸与辅因子吡哆醛5'-磷酸形成希夫碱，提供了对酶的底物特异性和催化机制的结构洞察，并暗示待裂解的羧基位于辅因子的si面。据我们所知，这可能是第一个报道的 DAPDC 外用醛亚胺。此外，HpDAPDC 的活性位点环处于“向下”构象并保护配体免受溶剂的影响。环中 Ile(148) 的突变极大地损害了催化效率。结合I148L突变体的结构分析，我们假设HpDAPDC采用诱导拟合催化机制，其中该循环通过“向下”和“向上”构象分别循环以稳定中间体和释放产物。我们的工作有望为设计 DAPDC 特异性抑制剂提供线索。
The meso-diaminopimelate decarboxylase (DAPDC, EC 4.1.1.20) catalyzes the final step of L-lysine biosynthesis in bacteria and is regarded as a target for the discovery of antibiotics. Here we report the 2.3 angstrom crystal structure of DAPDC from Helicobacter pylori (HpDAPDC). The structure, in which the product L-lysine forms a Schiff base with the cofactor pyridoxal 5'-phosphate, provides structural insight into the substrate specificity and catalytic mechanism of the enzyme, and implies that the carboxyl to be cleaved locates at the si face of the cofactor. To our knowledge, this might be the first reported external aldimine of DAPDC. Moreover, the active site loop of HpDAPDC is in a "down" conformation and shields the ligand from solvent. Mutations of Ile(148) from the loop greatly impaired the catalytic efficiency. Combining the structural analysis of the I148L mutant, we hypothesize that HpDAPDC adopts an induced-fit catalytic mechanism in which this loop cycles through "down" and "up" conformations to stabilize intermediates and release product, respectively. Our work is expected to provide clues for designing specific inhibitors of DAPDC.