Role of Phe-99 and Trp-196 of sepiapterin reductase from Chlorobium tepidum in the production of L-threo-tetrahydrobiopterin  

作　　者：Supangat Sun Ok Park Kyung Hye Seo Sang Yeol Lee Young Shik Park Kon Ho Lee 

机构地区：[1]Division of Applied Life Science (BK21 Program), Gyeongsang National University, Jinju 660-701, Korea [2]Plant Molecular Biology and Biotechnology Research Center, Gyeongsang National University, Jinju 660-701, Korea [3]Environmental Biotechnology National Core Research Center, Gyeongsang National University, Jinju 660-701, Korea [4]Mitochondrial Research Group, School of Biotechnology and Biomedical Science, Inje University, Kimhae 621-749, Korea

出　　处：《Acta Biochimica et Biophysica Sinica》2008年第6期513-518,共6页生物化学与生物物理学报（英文版）

摘　　要：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 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.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 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.

关 键 词：Chlorobium tepidum TETRAHYDROBIOPTERIN sepiapterin reductase site-directed mutagenesis ENZYME 

分 类 号：Q55[生物学—生物化学]