Isolation and Characterization of Sepiapterin Reductase from Drosophila melanogaster

Abstract

Summary Sepiapterin reductase, an enzyme that catalyses the synthesis of tetrahydrobiopterin (BH4). was partially purified from Drosophila melanogaster using ammonium sulfate fractionation. Affi-gel blue chromatography and hydroxyapatite chromatography. The molecular weight of the enzyme determined by Ultrogel AcA44 column was 39,000. When the enzyme was subjected to polyacrylamide gel electrophoresis in SDS. a 38.000 MW species was found to be the major protein band. The Km values for sepiapterin and NADPH were determined to he 75.4 µM. and 14 µM, respectively. The optimal temperature and pH for the reaction were 30°C and pH 5.7-6.7. The half-life of the activity was 30 minutes when treated at 48°C The enzyme was markedly inhibited by tri-and tetravalent cations. Fe3+ . Sn4+ and divalent cation. Cd2+ . It was found that pyrimidodiazcpine (a homopterin) and 2.4-diamino-6.7-diisopropyl caused the reduction of sepiapterin reductase activity by about 50% at the concentration of 0.1 mM. Among the neurotransmitters and their precursors tested. 3 mM concentrations of melatonin and N-acetylserotonin inhibited the enzyme activity completely. In addition to sepiapterin. the enzyme uses rather broad spectrum of carbonyl compounds as substrate including menadione. p-nitrohenzaldehyde, and various dicarhonyl compounds