Open AccessRole of Phe‐99 and Trp‐196 of sepiapterin reductase from Chlorobium tepidum in the production of L ‐threo‐tetrahydrobiopterin
Author(s) -
Park Sun Ok,
Seo Kyung Hye,
Lee Sang Yeol,
Park Young Shik,
Lee Kon Ho
Publication year - 2008
Publication title -
acta biochimica et biophysica sinica
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.771
H-Index - 57
eISSN - 1745-7270
pISSN - 1672-9145
DOI - 10.1111/j.1745-7270.2008.00422.x
Subject(s) - tetrahydrobiopterin , biochemistry , mutant , stereochemistry , alanine , substrate (aquarium) , chemistry , mutagenesis , allosteric regulation , enzyme , active site , biology , microbiology and biotechnology , amino acid , cofactor , gene , ecology
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° 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 K m values than the wild type. Remarkably, the W196A mutation resulted in a higher K m increase compared with the F99A mutation. Consistent with the results, the melting temperature ( T m ) 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.