Crystal Structure of Pyridoxamine-Pyruvate Aminotransferase from Mesorhizobium loti MAFF303099*
- Yu Yoshikane‡,
- Nana Yokochi‡,
- Masayuki Yamasaki§,
- Kimihiko Mizutani§,
- Kouhei Ohnishi¶,
- Bunzo Mikami§,
- Hideyuki Hayashi∥ and
- Toshiharu Yagi‡,1
- ‡Department of Bioresources Science, Faculty of Agriculture, Kochi University, Monobe-Otsu 200, Nankoku, Kochi 783-8502, Japan, the §Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University, Gokasho, Uji, Kyoto 611-0011, Japan, the ¶Research Institute of Molecular Genetics, Kochi University, Monobe-Otsu 200, Nankoku, Kochi 783-8502, Japan, and the ∥Department of Biochemistry, Osaka Medical College, 2-7 Daigakumachi, Takatsuki, Osaka 569-8686, Japan
- ↵1 To whom correspondence should be addressed. Tel. and Fax: 81-88-864-5191; E-mail: yagito{at}cc.kochi-u.ac.jp.
Abstract
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.
- Received September 27, 2007.
- Revision received October 31, 2007.
- The American Society for Biochemistry and Molecular Biology, Inc.
