Substrate recognition mechanism of L-lactate oxidase from Aerococcus viridans at 2.0Å resolution

Research Reactor Institute, Kyoto University, Japan* Institute for Enzyme Research, University of Tokushima, Japan** Fine Chemicals & Diagnostics Division, Asahi Kasei Pharma, Japan*** Institute for Protein Research, Osaka University, Japan****
○Yasufumi Umena* Kazuko Yorita** Takeshi Matsuoka*** Akiko Kita* Kiyoshi Fukui** Tomitake Tsukihara**** Yukio Morimoto*

L-lactate oxidase (LOX) from Aerococcus viridans belongs to the α-hydroxyacid oxidizing (α-HAO) flavoprotein family. LOX catalyzes the oxidization of L-lactate to pyruvate by abstracting two hydrogen atoms. We determined the crystal structure of wild type LOX to 2Å resolution. This molecule forms the tetramer possessing a circular four-fold symmetry. Each monomer consists of a typical α8/β8 TIM barrel structure and carries one FMN molecule as a cofactor bound at the carboxyl end of β strand barrel. The region around the N5 atom of FMN isoalloxazine ring on the si-side is accessible to the solvent in the catalytic site. Two arginine, Arg-181 and Arg-268, and one histidine, His-265, are conserved in the family and located above the si-side of FMN molecule in the active site. In this study, we carried out three mutants, R181M, R268M and H265Q, where the positively charged amino acid residues were replaced to non-charged ones, methionine and glutamine. These mutants showed dramatic decrease in the enzyme catalytic activity, and suggested that these residues, His-265, Arg-181 and Arg-268, are important for α-HAO catalytic mechanism. In this report, we also determined the crystal structures of these mutants with 2 Å resolution. We suggested for the role of these residues in catalytic activity from the structures that both Arg-181 and Arg-268 may work for the binding of substrates and His-265 is the catalyst for abstracting a proton from α-hydroxyacid. We also discuss the accessibility of the substrates to the active site by the location of water molecules and other structural feature.