Biosynthesis of Riboflavin: Structure and Properties of 2,5-Diamino-6-ribosylamino-4(3 H)-pyrimidinone 5′-phosphate Reductase of Methanocaldococcus jannaschii

Abstract

The pyrimidine reductase of the riboflavin biosynthetic pathway (MjaRED) specified by the open reading frame MJ0671 of Methanocaldococcus jannaschii was expressed in Escherichia coli using a synthetic gene. The synthetic open reading frame that was optimized for expression in E. coli directed the synthesis of abundant amounts of the enzyme with an apparent subunit mass of 25 kDa. The enzyme was purified to apparent homogeneity and was shown to catalyze the conversion of 2,5-diamino-6-ribosylamino-4(3 H)-pyrimidinone 5′-phosphate into 2,5-diamino-6-ribitylamino-4(3 H)-pyrimidinone 5′-phosphate at a rate of 0.8 μmol min −1 mg −1 at pH 8.0 and at 30 °C. The protein is a homodimer as shown by sedimentation equilibrium analysis and sediments at an apparent velocity of 3.5 S. The structure of the enzyme in complex with the cofactor nicotinamide adenine dinucleotide phosphate was determined by X-ray crystallography at a resolution of 2.5 Å. The folding pattern resembles that of dihydrofolate reductase with the Thermotoga maritima ortholog as the most similar structure. The substrate, 2,5-diamino-6-ribosylamino-4(3 H)-pyrimidinone 5′-phosphate, was modeled into the putative active site. The model suggests the transfer of the pro-R hydrogen of C-4 of NADPH to C-1′ of the substrate.