Sepiapterin reductase producing L-threo-dihydrobiopterin from Chlorobium tepidum.
A novel type of NADPH-dependent sepiapterin reductase, which catalysed uniquely the reduction of sepiapterin to l-threo-dihydrobiopterin, was purified 533-fold from the cytosolic fraction of Chlorobium tepidum, with an overall yield of 3%. The native enzyme had a molecular mass of 55 kDa and SDS/PAGE revealed that the enzyme consists of two subunits with a molecular mass of 26 kDa. The enzyme was optimally active at pH8.8 and 50 degrees C. Apparent Km values for sepiapterin and NADPH were 21 and 6.2 microM, respectively, and the kcat value was 5.0 s-1. Diacetyl could also serve as a substrate, with a Km of 4.0 mM. The inhibitory effects of N-acetylserotonin, N-acetyldopamine and melatonin were very weak. The Ki value of N-acetyldopamine was measured as 400 microM. The N-terminal amino acid sequence was revealed as Met-Lys-His-Ile-Leu-Leu-Ile-Thr-Gly-Ala-Xaa-Lys - Lys - Ile - Xaa - Arg - Ala - Ile - Ala - Leu - Glu - Xaa - Ala - Arg - Xaa-Xaa-Xaa-His-His-His-, which shared relatively high sequence similarity with other sepiapterin reductases.
Cho
S H
SH
Laboratory of Biophysics, Department of Microbiology, College of Natural Sciences, and Research Center for Molecular Microbiology, Seoul National University, Seoul 151-742, Republic of Korea.
Na
J U
JU
Youn
H
H
Hwang
C S
CS
Lee
C H
CH
Kang
S O
SO
D000429:Alcohol Oxidoreductases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D001708:Biopterins / Q000031:analogs & derivatives* / Q000096:biosynthesis; D002384:Catalysis; D019414:Chlorobi / Q000201:enzymology* / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D013696:Temperature
D004791:Enzyme Inhibitors; D001708:Biopterins; C017226:7,8-dihydrobiopterin; D000429:Alcohol Oxidoreductases; C022630:sepiapterin reductase