Role of Phe-99 and Trp-196 of sepiapterin reductase from Chlorobium tepidum in the production of L-threo-tetrahydrobiopterin.
Sepiapterin reductase from Chlorobium tepidum (cSR) catalyzes the synthesis of a distinct tetrahydrobiopterin (BH4), L-threo-BH4, different from the mammalian enzyme product. The 3-D crystal structure of cSR has revealed that the product configuration is determined solely by the substrate binding mode within the well-conserved catalytic triads. In cSR, the sepiapterin is stacked between two aromatic side chains of Phe-99 and Trp-196 and rotated approximately 180 degrees C around the active site from the position in mouse sepiapterin reductase. To confirm their roles in substrate binding, we mutated Phe-99 and/or Trp-196 to alanine (F99A, W196A) by site-directed mutagenesis and comparatively examined substrate binding of the purified proteins by kinetics analysis and differential scanning calorimetry. These mutants had higher Km values than the wild type. Remarkably, the W196A mutation resulted in a higher Km increase compared with the F99A mutation. Consistent with the results, the melting temperature (Tm) in the presence of sepiapterin was lower in the mutant proteins and the worst was W196A. These findings indicate that the two residues are indispensable for substrate binding in cSR, and Trp-196 is more important than Phe-99 for different stereoisomer production.
Division of Applied Life Science, BK21 Program, Gyeongsang National University, Jinju 660-701, Korea.
Supangat
S
Supangat
Sun Ok
SO
Park
Kyung Hye
KH
Seo
Sang Yeol
SY
Lee
Young Shik
YS
Park
Kon Ho
KH
Lee
D000429:Alcohol Oxidoreductases / Q000737:chemistry* / Q000235:genetics; D000595:Amino Acid Sequence; D001708:Biopterins / Q000031:analogs & derivatives* / Q000138:chemical synthesis; D041883:Chlorobium / Q000201:enzymology*; D003198:Computer Simulation; D004789:Enzyme Activation; D004795:Enzyme Stability; D008956:Models, Chemical; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010649:Phenylalanine / Q000737:chemistry*; D013329:Structure-Activity Relationship; D014364:Tryptophan / Q000737:chemistry*
D001708:Biopterins; D010649:Phenylalanine; D014364:Tryptophan; D000429:Alcohol Oxidoreductases; C022630:sepiapterin reductase; C003402:sapropterin