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Functionally important residues tyrosine-171 and serine-158 in sepiapterin reductase
- Source :
- Biochimica et Biophysica Acta (BBA) - Protein Structure and Molecular Enzymology. 1431:306-314
- Publication Year :
- 1999
- Publisher :
- Elsevier BV, 1999.
-
Abstract
- 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.
- Subjects :
- Sepiapterin
DNA, Complementary
Stereochemistry
Biophysics
Dehydrogenase
Reductase
Biochemistry
Cofactor
chemistry.chemical_compound
Structural Biology
Escherichia coli
Serine
medicine
Animals
Humans
Cloning, Molecular
Pterin
Sepiapterin reductase
Molecular Biology
Binding Sites
biology
Active site
Tetrahydrobiopterin
Biopterin
Rats
Alcohol Oxidoreductases
chemistry
Mutation
Mutagenesis, Site-Directed
biology.protein
Tyrosine
medicine.drug
Subjects
Details
- ISSN :
- 01674838
- Volume :
- 1431
- Database :
- OpenAIRE
- Journal :
- Biochimica et Biophysica Acta (BBA) - Protein Structure and Molecular Enzymology
- Accession number :
- edsair.doi.dedup.....ef493f3775c9c247c5b5b8825fa5eb53