Kenji Aki

Division of Functional Food Chemistry

In order to clearify the relationship between structure and function of flavoenzyme, the present study is to focus on the the analysis of similarities and differences in structure and function of the family group of flavoenzymes.
On the other hand, the physiological study on the oxygen metabolism is progress to elucidate the mechanisms of generation of reactive oxygen and nitric oxide by some flavoenzymes.

Research Projects as follows
(1) Structure and function of flavoenzymes ,glutathione reductase and lipoamide dehydrogenase or L-lactate oxidase and L-lactate monooxygenase
(2) Enzymatic generation and biological functions of reactive oxygen species and moreover biological antioxidant actions

Research Summary as follows
(1) Properties of L-lactate oxidase from Aerococcus viridans are described. The gene encoding the enzyme has been isolated. From its cDNA sequence the amino acid sequence has been derived and shown to have high similarity with those of other enzymes catalyzing oxidation of L-a-hydroxy acids, including flavocytochrome b2, lactate monooxygenase, glycolate oxidase, mandelate dehydrogenases and a long chain a-hydroxy acid oxidase. The enzyme is a flavoprotein containing FMN as prosthetic group. It shares many properties of other a-hydroxy acid oxidizing enzymes, eg stabilization of the anionic semiquinone form of the flavin, facile formation of flavin-N(5)-sulfite adducts and a set of conserved amino acid residues around the bound flavin. Steady-state- and rapid reaction kinetics of the enzyme have been studied and found to share many characteristics with those of L-lactate monooxygenase, but to differ from the latter in quantitative aspects. It is these quantitative differences between the two enzymes which account for the differences in the overall reactions catalyzed. These differences arise from different stabilities of a common intermediate of reduced flavin enzyme and pyruvate. In the case of the monooxygenase this complex is very stable, and is the form that reacts with O2 to give a complex in which the oxidative decarboxylation occurs, yielding the products, acetate, CO2, and H2O. With lactate oxidase, the complex dissociates rapidly, with the result that it is the free reduced flavin form of the enzyme that reacts with O2, to give the observed products, pyruvate and H2O2. The mutagenetic study of L-lactate oxidase is progressing.

(2) The oxydase reaction of lipoamide dehydrogenase with NADH generates superoxide radicals and hydrogen peroxide under aerobic conditions. ESR spin trapping using 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) was applied to characterize the oxygen radical species generated by lipoamide dehydrogenase and the mechanism of their generation. During the oxidase reaction of lipoamide dehydrogenase, DMPO-OOH and DMPO-OH signals were observed. The DMPO-OOH signal disappeared on addition of superoxide dismutase. These results demonstrate that the DMPO-OOH adduct was produced from the superoxide radical generated by lipoamide dehydrogenase. In the presence of dimethyl sulfoxide, a DMPO-CH3 signal appeared at the expense of the DMPO-OH signal, indicating that the DMPO-OH adduct was produced directly from the hydroxyl radical rather than by decomposition of the DMPO-OOH adduct. The DMPO-OH signal decreased on addition of superoxide dismutase, catalase, or diethylenetriaminepentaacetic acid, indicating that the hydroxyl radical was generated via the metal-catalyzed Haber-Weiss reaction from the superoxide radical and hydrogen peroxide. Addition of ferritin to the NADH-lipoamide dehydrogenase system resulted in a decrease of the DMPO-OOH signal, indicating that the superoxide radical interacted with ferritin iron. The mechanism of reductive mobilization of iron from ferritin by lipoamide dehydrogenase is studying.

Selected Papers;
1) Tsuge H., Kawakami R., Sakuraba H., Ago H., Miyano M., Aki K., Katunuma N. and Ohshima T.: Crystal Structure of a Novel FAD-, FMN-, and ATP-containing L-Proline Dehydrogenase Complex from Pyrococcus horikoshi. J. Biol. Chem. 2005;280(35):31045-31049.

2) Mitsui T., Kawajiri M., Kunisige M., Endo T., Akaike M., Aki K. and Matsumoto T.: Functional Association between Nicotinic Acetylcholine Receptor and Sarcomeric Proteins via Action and Desmine Filaments. J. Cell Biochem. 2000;77(4):584-595.

3) Yorita K., Janko K., Aki K., Ghisia S., Palfey B.A., Misaki H. and Massey V.: On the Reaction Mechanism of L-Lactate Oxidase: Quantitative Structure-Activity Analysis of the Reaction with Para-substituted L-Mandelates. Proc. Natl. Acd. Sci. USA. 1997;94(18):9590-9595.

4) Yorita K., Aki K., Ohkuma-Soejima T., Kokubo T., Misaki H. and Massey V.: Conversion of L-Lactate Oxidase to a Long Chain L-Hydroxyacid Oxidase by Site-directed Mutagenesis of Alanine 95 to Glysine. J. Biol. Chem. 1996;271(45):28300-28305.

5) Maeda-Yorita K., Aki K., Sagai H., Misaki H. and Massey V.: L-lactate oxidase and L-lactate monooxygenase: Mechanistic variations on a common structural theme. Biochemie. 1995;77:631-642.
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