Characterization of recombinant Dictyostelium discoideum sepiapterin reductase expressed in E-coli

A cDNA clone (SSC801) putatively encoding sepiapterin reductase (SR) was ob tained from the expressed sequence tag clones of Dictyostelium discoideum, The cDNA sequence of 878 nucleotides constituted an ORF of 265 amino acid r esidues but was missing a few N-terminal residues, The deduced amino acid s equence showed 29.8% identity with mouse SR sequence and a molecular mass o f 29,969 Da, The coding sequence was cloned in E. coli expression vector an d overexpressed, The purified His-tag recombinant enzyme was confirmed to h ave the genuine activity of SR to produce tetrahydrobiopterin from 6-pyruvo yltetrahydropterin in a coupled assay with 6-pyruvoyltetrahydropterin synth ase as well as dihydrobiopterin from sepiapterin. However, dictyopterin was not observed in our assay condition. The enzyme was also inhibited by N-ac etylserotonin and to a lesser extent by melatonin, K-m values for NADPH and sepiapterin were 51.8 +/- 2.7 mu M and 40 +/- 2 mu M, respectively. V-max was determined as 0.14 mu mol/min/mg of protein.