Crystal Structure of Pyridoxamine-Pyruvate Aminotransferase from Mesorhizobium loti MAFF303099*
Crystal Structure of Pyridoxamine-Pyruvate Aminotransferase from Mesorhizobium loti MAFF303099*
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DOI：
10.1074/jbc.m708061200
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发表时间：
2008-01
影响因子：
4.8
通讯作者：
Yu Yoshikane;N. Yokochi;M. Yamasaki;K. Mizutani;K. Ohnishi;B. Mikami;H. Hayashi;T. Yagi
中科院分区：
文献类型：
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作者：
Yu Yoshikane;N. Yokochi;M. Yamasaki;K. Mizutani;K. Ohnishi;B. Mikami;H. Hayashi;T. Yagi
吡哆胺-丙酮酸氨基转移酶（PPAT; EC 2.6.1.30）是吡哆醛5′-磷酸非依赖性氨基转移酶，催化吡哆胺和丙酮酸之间的可逆转氨作用形成吡哆醛和l-丙氨酸。在2.0A的解析度下，PPAT的空间群为P43212，R因子为15.6%（Rfree = 20.6%）。此外，PPAT与吡哆胺、吡哆醛和吡哆酰-1-丙氨酸的复合物的结构已分别在1.7、1.7和2.0 A的分辨率下被细化至15.6、15.4和14.5%的R因子（Rfree = 18.6、18.1和18.4%）。PPAT是一种同源四聚体，每个亚基由一个大的N-末端结构域和一个较小的C-末端结构域组成，N-末端结构域由7个β-折叠和8个α-螺旋组成，C-末端结构域由3个β-折叠和4个α-螺旋组成。底物吡哆醛通过醛亚胺键与活性位点中的Lys-197结合。底物氨基/酮酸的α-羧酸酯基团与Arg-336和Arg-345形成氢键。结构表明Glu-68的大侧链干扰了吡哆醛5′-磷酸的磷酸部分的结合，并使PPAT对吡哆醛具有特异性。根据酶的结构和动力学研究结果，探讨了酶的反应机理。
Pyridoxamine-pyruvate aminotransferase (PPAT; EC 2.6.1.30) is a pyridoxal 5′-phosphate-independent aminotransferase and catalyzes reversible transamination between pyridoxamine and pyruvate to form pyridoxal and l-alanine. The crystal structure of PPAT from Mesorhizobium loti has been solved in space group P43212 and was refined to an R factor of 15.6% (Rfree = 20.6%) at 2.0Å resolution. In addition, the structures of PPAT in complexes with pyridoxamine, pyridoxal, and pyridoxyl-l-alanine have been refined to R factors of 15.6, 15.4, and 14.5% (Rfree = 18.6, 18.1, and 18.4%) at 1.7, 1.7, and 2.0Å resolution, respectively. PPAT is a homotetramer and each subunit is composed of a large N-terminal domain, consisting of seven β-sheets and eight α-helices, and a smaller C-terminal domain, consisting of three β-sheets and four α-helices. The substrate pyridoxal is bound through an aldimine linkage to Lys-197 in the active site. The α-carboxylate group of the substrate amino/keto acid is hydrogen-bonded to Arg-336 and Arg-345. The structures revealed that the bulky side chain of Glu-68 interfered with the binding of the phosphate moiety of pyridoxal 5′-phosphate and made PPAT specific to pyridoxal. The reaction mechanism of the enzyme is discussed based on the structures and kinetics results.