Two Crystal Structures of Rat Liver Dyhydropteridine Reductase

Abstract

Dihydropteridine reductase (DHPR EC 1.6.99.10) catalyzes the reduction of quinonoid dihydrobiopterin(qBH2) to tetrahydrobiopterin, using NADH as a cofactor. Tetrahydrobiopterin is a cofactor for the hydroxylating enzymes -phenylalanine hydroxylase, tyrosine hydroxylase and phenylalanine hydroxylase. The genetic errors in phenylalanine hydroxylase, DHPR or in genes coding for enzymes on the biosynthetic route from GTP to qBH2, the substrate for DHPR, together contribute to the inherited disease phenylketonuria (PKU). Recently several of the errors occurring in DHPR have been identified. The structure of DHPR complexed with its cofactor, NADH in an orthorhombic form (C2221) has been solved and refined with 2.0 A resolution data. In addition we determined the crystal structure of a monoclinic form which has two dimers in the asymmetric unit. The orthorhombic form has only one monomer per asymmetric unit. Modelling studies suggested the involvment of Trp 86 and Tyr 146 in substrate binding. The W86I mutant was therefore constructed and was found to have diminished enzymatic activity. The Y146F mutant was constructed and was found to have no activity at all.Dihydropteridine reductase (DHPR EC 1.6.99.10) catalyzes the reduction of quinonoid dihydrobiopterin(qBH2) to tetrahydrobiopterin, using NADH as a cofactor. Tetrahydrobiopterin is a cofactor for the hydroxylating enzymes -phenylalanine hydroxylase, tyrosine hydroxylase and phenylalanine hydroxylase. The genetic errors in phenylalanine hydroxylase, DHPR or in genes coding for enzymes on the biosynthetic route from GTP to qBH2, the substrate for DHPR, together contribute to the inherited disease phenylketonuria (PKU). Recently several of the errors occurring in DHPR have been identified. The structure of DHPR complexed with its cofactor, NADH in an orthorhombic form (C2221) has been solved and refined with 2.0 A resolution data. In addition we determined the crystal structure of a monoclinic form which has two dimers in the asymmetric unit. The orthorhombic form has only one monomer per asymmetric unit. Modelling studies suggested the involvment of Trp 86 and Tyr 146 in substrate binding. The W86I mutant was therefore constructed and was found to have diminished enzymatic activity. The Y146F mutant was constructed and was found to have no activity at all.