Site-Directed Mutagenesis of Residues in the Active Site of Sepiapterin Reductase

Abstract

Tetrahydrobiopterin (BH₄) is the natural cofactor for producing neurotransmitters such as catecholamines, indoleamines, and nitric oxide. Sepiapterin reductase (SPR; EC 1. 1. 1. 153) catalyzes the terminal step in the biosynthesis of BH4 and is regulated by phosphorylation and feedback inhibitors such as N-acetyl serotonin (NAS). SPR reduces the two carbonyl groups (-CO-CO-) of 6-pyruvoyl-tetrahydropterin to dihydroxy groups (-CHOH-CHOH-) to form BH₄, and it also reduces sepiapterin (6-1′-oxo-2′-hydroxypropyl-7,8-dihydropterin) to dihydrobiopterin (6-1′,2′-dihydroxypropyl-7,8-dihydropterin). SPR shows a high specificity for the carbonyl groups of these pteridines, nevertheless, SPR can catalyze the NADPH-dependent reduction of various non-pteridine carbonyl compounds such as phenylpropanedione like many other enzymes of the NADP(H)-preferring short-chain dehydrogenase/reductase (SDR) family. In this study, we constructed various deletion and point mutants of SPR for residues postulated as the active site of SDR family enzymes and determined the sites of SPR for binding NADPH and substrate and the catalytic site.