Functionally important residues tyrosine-171 and serine-158 in sepiapterin reductase.
The active site of sepiapterin reductase (SPR), which is a member of the NADP(H)-preferring short-chain dehydrogenase/reductase (SDR) family and acts as the terminal enzyme in the biosynthetic pathway of tetrahydrobiopterin cofactor (BH4), was investigated by truncation and site-directed mutagenesis. The truncation mutants showed that N-terminal and C-terminal residues contribute to bind coenzyme and substrate, respectively. The mutant rSPRA29V showed decreased activity; however, the A-X-L-L-S sequence, which has been reported as a putative pterin binding site, was estimated to preferably work as a component in the region for binding coenzyme rather than substrate. Site-directed mutants of rSPRS158D, rSPRY171V, and rSPRK175I showed low, but significant, activity having similar Km values and kcat/Km values less than 25%, for both sepiapterin and NADPH. Both amino acids Tyr-171 and Ser-158 are located within a similar distance to the carbonyl group of the substrate in the crystal structure of mouse SPR, and the double point mutant rSPRY171V+S158D was indicated to be inactive. These results showed that Ser-158, Tyr-171, and Lys-175 contributed to the catalytic activity of SPR, and both Tyr-171 and Ser-158 are simultaneously necessary on proton transfer to the carbonyl functional groups of substrate.
Fujimoto
K
K
Department of Biochemistry, Meikai University School of Dentistry, Sakado, Saitama 350-0283, Japan. kengo@dent.meikai.ac.jp
Ichinose
H
H
Nagatsu
T
T
Nonaka
T
T
Mitsui
Y
Y
Katoh
S
S
D000429:Alcohol Oxidoreductases / Q000737:chemistry* / Q000235:genetics; D000818:Animals; D001665:Binding Sites; D001708:Biopterins / Q000031:analogs & derivatives / Q000096:biosynthesis; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000378:metabolism; D004926:Escherichia coli / Q000378:metabolism; D006801:Humans; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D051381:Rats; D012694:Serine / Q000737:chemistry; D014443:Tyrosine / Q000737:chemistry
D018076:DNA, Complementary; D001708:Biopterins; D014443:Tyrosine; D012694:Serine; D000429:Alcohol Oxidoreductases; C022630:sepiapterin reductase; C003402:sapropterin