New aspects in biopterin biosynthesis in man

Tetrahydrobiopterin is synthesized from guanosine triphosphate (GTP)in several enzymatic steps [1,2]. Details of the pathway of the biopterin biosynthesis in mammals are still unresolved [3]. In rat and human brain the rate-limiting enzyme was GTP cyclohydrolase [2], followed by D-erythro-7,8-dihydroneopterin triphosphate synthetase. D-Erythro-7,8-dihydroneopterin triphosphate was directly converted to 'quinonoid' dihydrobiopterin by an enzyme not requiring pyridine nucleotides or other cofactors for catalytic activity [2]. This is in contrast to other results [1] demonstrating that the conversion of D-erythro-7,8- dihydroneopterin triphosphate to L-erythro-dihydrobiopterin required 3 distinct protein fractions. A Mg2+ -dependent enzyme (A2) catalyzed the conversion of D-erythro-7,8-dihydroneopterin triphosphate to an intermediateX of unknown structure proposed to be 6(1 ',2'-dioxopropyl)-7,8-dihydropterin which could be degraded to pterin and pyruvic acid [1]. A heat labile and NADPH-dependent enzyme (A1) converted X to sepiapterin. We reported in [4] that 3'-hydroxysepiapterin is excreted in small amounts in the urine of healthy individuals but it is markedly increased in patients with dihydrobiopterin-deficiency. In connection with our studies of atypical phenylketonuria, it was of interest to investigate the biopterin biosynthesis in man. These data indicate that, indeed, also in human kidney and liver, biopterin synthesis might proceed via compound X and sepiapterin

Purification of 6-pyruvoyl-tetrahydropterin synthase from human liver

The enzyme which catalyzes the first step in the conversion of dihydroneopterin triphosphate to tetrahydrobiopterin has been purified approx. 40,000-fold from human liver to apparent homogeneity. The enzyme has a native molecular weight of ~83,000 and consists of four identical subunits, each of which has a molecular weight of ~19,000. It contains carbohydrates and is remarkably stable to heat treatment. In the presence of purified sepiapterin reductase, Mg2+, and NADPH, this enzyme catalyzes efficiently the formation of tetrahydrobiopterin from dihydroneopterin triphosphate. This indicates that these two proteins are sufficient for the overall conversion

Purification and characterization of a carbonyl-reductase from human liver, which is competent in the reduction of 6-pyruvoyl-tetrahydropterin

An enzyme which reduces 6-pyruvoyl-tetrahydropterin has been purified to apparent homogeneity from human liver. It consists of a single polypeptide chain with a molecular weight of 35 kDa, has an isoelectric point of 5.9 ± 0.1 and contains no glycosyl residues. The pure enzyme has a specific activity of 450 mU/mg protein at pH 7.0 in 10 mM potassium phosphate buffer. It converts 6-pyruvoyl-tetrahydropterin to 6-lactoyltetrahydropterin by transfer of the pro 4R-hydrogen of NADPH to form the side chain -OH at position C(2') of the substrate. Km values are 1.8 μM for 6-pyruvoyl-tetrahydropterin and 5.5 μM for NADPH. Polyclonal antibodies raised against the purified enzyme recognize 6-pyruvoyl-tetrahydropterin reductase in Western blot and ELISA but do not cross-react with human sepiapterin reductase. The enzyme appears to be identical with aldose reductase

Pterin-4a-carbinolamine dehydratase from pseudomonas aeruginosa : characterization, catalytic mechanism and comparison to the human enzyme

The three-dimensional structure of pterin-4a-carbinolamine dehydratase (PCD) from Pseudomonas aeruginosa has been solved. Based on this we have investigated the roles of putative active center residues through functional replacement by site-directed mutagenesis. Three histidines, His73, His74 and His91, appear to be involved in dehydration catalysis. The three-dimensional positions of these residues match those of corresponding histidines at the active center of human PCD. Based on the coincidence of catalytic parameters, and on the similar effects induced by the mutations, it is concluded that the substrate binding mode and the reaction mechanisms of bacterial and human PCD are basically identical