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Journal of Biochemistry Advance Access originally published online on March 29, 2007
Journal of Biochemistry 2007 141(6):799-815; doi:10.1093/jb/mvm084
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© 2007 The Japanese Biochemical Society.

Kinetic Studies on the Role of Lys-171 and Lys-358 in the ß Subunit of Sarcosine Oxidase from Corynebacterium sp. U-96

Mutsumi Saito1, Miho Kanno1, Haruo Iizuka2 and Haruo Suzuki1,2,*

1Division of Bioscience, Graduate School of Basic Life Science; and 2Department of Biosciences, School of Science, Kitasato University, Kitasato 1-15-1, Sagamihara, Kanagawa 228-8555, Japan

*To whom correspondence should be addressed. Tel: +81-42-778-9410, Fax: +81-42-778-9942, E-mail: suzukih{at}kitasato-u.ac.jp

Received January 30, 2007; Accepted March 21, 2007


  Abstract

Heterotetrameric sarcosine oxidase from Corynebacterium sp.U-96(SO-U96) contains non-covalent and covalent flavins. Lys-358 and Lys-171 in the ß subunit is present at non-covalent flavin adenine dinucleotide (FAD)- and covalent flavin monodinucleotide (FMN)-binding sites, respectively. The Lys-358 mutant, K358R showed 0.07% activity and higher apparent Km for sarcosine than the wild-type enzyme, but K358A and K358D mutants showed no activity, suggesting the importance of amino group of Lys358 in the sarcosine-binding to the enzyme. The Lys171 mutants, K171R, K171A and K171D showed 58, 39 and 32% activity of the wild-type enzyme, respectively. An apparent Km for oxygen and Kd of enzyme–sulphite complex increased by the mutation. The rate of reduction of the FAD of K171 mutants with sarcosine did not change by the mutation. The stopped-flow photodiode array analyses of the anaerobic reduction with sarcosin of the wild-type and K171 mutant enzymes showed characteristic spectra of neutral and anionic semiquinones, especially for K171A enzyme. On the basis of these results, the reductive-half reaction of the wild-type and K171 mutant enzymes is explained by a mechanism involving the semiquinones. Low activity of K171 mutants is suggested to be derived from the low rate of oxidation of the reduced FMN in the enzyme.

Key Words: essential lys, flavoenzyme, kinetic mechanism, sarcosine oxidase, site-directed mutagenesis

Abbreviations: CPR, cytochrome P450 reductase; EDTA, ethylenediaminetetraacetic acid; FAD, flavin adenine dinucleotide; FMN, flavin mononucleotide; IAM, iodeacetamide; IPTG, isopropyl-ß-d-thiogalactopyranoside; NAD+, nicotinamide adenine dinucleotide, oxidized form; PDH, L-proline dehydrogenase; Sar, sarcosine; SO, sarcosine oxidase; SO-ART, SO from Arthrobacter sp.1-IN; SO-MAL, SO from Pseudomonas maltophilia; SDS-PAGE, sodium dodecylsulphate polyacrylamide gel electrophoresis; SO-PAO, SO from Pseudomonas aeruginosa PAO1; SO-P1, SO from Corynebacterium sp.P-1; SO-U96, SO from Corynebacterium sp. U-96


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