Purification and physicochemical properties of NADPH-specific dihydropteridine reductase from bovine and human livers

A new type of dihydropteridine reductase [EC 1.6.99.10], which is specific for NADPH as the substrate in the reduction of quinonoid-dihydropterin to tetrahydropterin, was purified to homogeneity from bovine liver and human liver. The molecular weight of the enzyme was determined to be 65,000-70,000. The enzyme was composed of two subunits with identical molecular weight of 35,000; the amino terminal residue was determined to be valine. The isoelectric point of the enzyme was 7.05. The physicochemical properties of this enzyme were quite different from those of bovine liver NADH-specific dihydropteridine reductase [EC 1.6.99.7]. NADPH-specific dihydropteridine reductase did not cross-react with an antiserum raised against the NADH-specific dihydropteridine reductase, nor did the latter enzyme react with an antiserum to the former enzyme, indicating that the two enzymes have no common antigenic determinants. NADPH-specific dihydropteridine reductase from human liver was shown to have properties similar to those of the bovine liver enzyme.     

Inhibition of dihydropteridine reductase in rat striatal synaptosomes and from human liver by metabolites of biogenic amines

Catecholamines are potent noncompetitive inhibitors of dihydropteridine reductase in rat striatal synaptosomal preparations or purified from human liver. Their metabolites, except homovanillic acid, also inhibit the enzyme from both sources. The inhibitory potency of these compounds depends on the presence of the catechol or the 4-hydroxyphenyl structure, but may be modified by the 2-carbon side chain and its substituents. Indoleamines which have a hydroxylated aromatic nucleus (5-hydroxytryptamine and 5,6-dihydroxytryptamine) are equally inhibitory to the enzyme. These results suggest that biogenic amines themselves rather than their metabolites may serve as physiological inhibitors of dihydropteridine reductase in rat brain.     

Purification and properties of NADH-specific dihydropteridine reductase from human erythrocytes

NADH-specific dihydropteridine reductase (EC 1.6.99.7) has been purified from human erythrocytes in essentially homogeneous form. The molecular weight of the enzyme was estimated to be 46,000 by Sephadex G-100 gel filtration. The enzyme reacted with antiserum against NADH-specific dihydropteridine reductase from bovine liver and formed a single immunoprecipitin line in the Ouchterlony double-diffusion system. This precipitin line completely fused with that formed between the human liver enzyme and the antiserum. With use of 5,6,7,8-tetrahydro-6-methylpterin, Km values of the erythrocyte enzyme for NADH and NADPH were determined to be 0.94 and 47 mumol/l, respectively. Vmax values were 58.7 mumol/min/mg with NADH and 6.41 mumol/min/mg with NADPH. The average activity of NADH-specific dihydropteridine reductase of 9 human blood samples from healthy males (20-25 years old) was calculated to be approximately 600 mU/g of hemoglobin, 1.8 mU per 20 microliters of blood, or 1.9 mU per 10(8) erythrocytes.     

Physarum polycephalum expresses a dihydropteridine reductase with selectivity for pterin substrates with a 6-(1', 2'-dihydroxypropyl) substitution

Physarum polycephalum is one of few non-animal organisms capable of synthesizing tetrahydrobiopterin from GTP. Here we demonstrate developmentally regulated expression of quinoid dihydropteridine reductase (EC 1.6.99.7), an enzyme required for recycling 6,7-[8H]-dihydrobiopterin. Physarum also expresses phenylalanine-4-hydroxylase activity, an enzyme that depends on dihydropteridine reductase. The 24.4 kDa Physarum dihydropteridine reductase shares 43% amino acid identity with the human protein. A number of residues important for function of the mammalian enzyme are also conserved in the Physarum sequence. In comparison to sheep liver dihydropteridine reductase, purified recombinant Physarum dihydropteridine reductase prefers pterin substrates with a 6-(1', 2'-dihydroxypropyl) group. Our results demonstrate that Physarum synthesizes, utilizes and metabolizes tetrahydrobiopterin in a way hitherto thought to be restricted to the animal kingdom.     

A new enzyme, NADPH-dihydropteridine reductase in bovine liver.

An enzyme designated as NADPH-dihydropteridine reductase was found in the extract of bovine liver and partially purified. In contrast to NADH-dpendent dihydropteridine reductase [EC 1.6.99.7], the enzyme catalyzes the reduction of quinonid-dihydropterin to tetrahydropterin in the presence of NADPH. The two enzymes were separated by column chromatography on DEAE-sephadex. Tyrosine formation in the phenylalanine hydroxylation system was also stimulated by NADPH-dihydropteridine reductase. The existence of these two dihydropteridine reductases suggests that the tetrahydro from ofpteridine cofactor may be regenerated in two different ways in vivo.     

Genetics of the mammalian phenylalanine hydroxylase system. Studies of human liver phenylalanine hydroxylase subunit structure and of mutations in phenylketonuria.

Phenylalanine hydroxylase was purified from crude extracts of human livers which show enzyme activity by usine two different methods: (a) affinity chromatography and (b) immunoprecipitation with an antiserum against highly purified monkey liver phenylalanine hydroxylase. Purified human liver phenylalanine hydroxylase has an estimated mol. wt. of 275 000, and subunit mol. wts. of approx. 50 000 and 49 000. These two molecular-weight forms are designated H and L subunits. On two-dimensional polyacrylamide gel under dissociating conditions, enzyme purified by the two methods revealed at least six subunit species, which were resolved into two size classes. Two of these species have a molecular weight corresponding to that of the H subunit, whereas the other four have a molecular weight corresponding to that of the L subunit. This evidence indicates that active phenylalanine hydroxylase purified from human liver is composed of a mixture of sununits which are different in charge and size. None of the subunit species could be detected in crude extracts of livers from two patients with classical phenylketonuria by either the affinity or the immunoprecipitation method. However, they were present in liver from a patient with malignant hyperphenylalaninaemia with normal activity of dihydropteridine reductase.     

NADH-specific dihydropteridine reductase in mastocytoma P-815 cells

NADH-specific dihydropteridine reductase [EC 1.6.99.7] was purified from mouse mastocytoma P-815 cells. Km values for NADH and NADPH were determined to be 1.4 microM and 32 microM, respectively, using tetrahydro-6-methylpterin. Molecular weight was 50,000, and subunit molecular weight was 25,000. The enzymes from P-815 and liver of host mouse (DBA/2) showed similar electrophoretic mobility on polyacrylamide gel. The P-815 enzyme reacted with antiserum against bovine liver NADH-specific dihydropteridine reductase, forming a single precipitin line.     

Structural studies and isolation of cDNA clones providing the complete sequence of rat liver dihydropteridine reductase

The cleavage of reductively alkylated rat liver dihydropteridine reductase with cyanogen bromide afforded a mixture of peptides, six of which (CB-1 to CB-6) were isolated and purified by C8 reverse-phase high performance liquid chromatography. Portions of peptides CB-1, CB-4, and CB-6 were sequenced by automated Edman degradation and high performance liquid chromatography and the carboxyl-terminal region by conventional procedures. Further proteolytic digestion of CB-6 and isolation of the products afforded a seven-amino acid peptide. A low degeneracy probe comprising 20 nucleotides was synthesized from the sequence of this peptide and was used to screen a rat liver cDNA expression library constructed in the vector lambda gt 10. Positive clones were isolated, and detailed examination of five of these by restriction endonucleases and dideoxy sequence analyses allowed identification of the entire coding region for dihydropteridine reductase. The gene was found to code for a protein of 240 amino acids (excluding the methionine initiator) of Mr = 25,420. Each of the sequences corresponding to the peptides CB-1, CB-4, CB-6, and the carboxyl terminus were identified in the deduced protein sequence. The rat enzyme is highly homologous to the human dihydropteridine reductase; the two proteins differ in only 10 amino acids, and all are conservative substitutions. In contrast, the sequence shows little homology with that of mammalian dihydrofolate reductase: reduced pyridine nucleotide-requiring enzymes with superficial mechanistic similarities.     

Rat striatal synaptosomes as a model system for studying the inhibition of dihydropteridine reductase activity

The distribution of dihydropteridine reductase between soluble and particulate fractions in synaptosomes parallels that of lactate dehydrogenase, but not monoamine oxidase. Ki and I50 values for inhibitors obtained with the enzyme-rich P2 fraction and its twice-washed fraction (P2W2) were essentially the same, and were similar to those obtained with highly purified human liver enzyme. Dihydropteridine reductase inhibitory potency of multi-ring compounds containing a catechol-moiety was greater than that of single ring catecholic compounds, which in turn was greater than that of p-hydroxy-phenolic compounds. The P2 fraction of rat striatal synaptosomal preparations may serve as a convenient source of dihydropteridine reductase for studying the inhibition of this enzyme.     

Catalytic properties of NADPH-specific dihydropteridine reductase from bovine liver

The catalytic properties of a new type of dihydropteridine reductase, NADPH-specific dihydropteridine reductase [EC 1.6.99.10], from bovine liver, were studied and compared with those of the previously characterized enzyme, NADH-specific dihydropteridine reductase [EC 1.6.99.7]. With quinonoid-dihydro-6-methylpterin, approximate Km values of NADPH-specific dihydropteridine reductase for NADPH and NADH were estimated to be 1.4 micron and 2,900 microns, respectively. The Vmax values were 1.34 mumol/min/mg with NADPH and 1.02 mumol/min/mg with NADPH. With NADPH, the Km values of the enzyme for the quinonoid-dihydro forms of 6-methylpterin and biopterin were 1.4 micron and 6.8 microns, respectively. The enzyme was inhibited by its reaction product, NADP+, in a competitive manner, and the inhibition constant was determined to be 3.2 microns. The enzyme was severely inhibited by L-thyroxine and by 2,6-dichlorophenolindophenol.     

Isolation and characterization of dihydropteridine reductase from Pseudomonas species.

Dihydropteridine reductase isolated from the bacterium Pseudomonas species (ATCC 11299a) has been purified approximately 450-fold byammonium sulfate precipitation and diethylaminoethyl-cellulose chromatographic procedures. The preparation is at least 80% pure as judged by polyacrylamide gels. Its molecular weight was determined to be about 44,000. Both dihydropteridine reductase and phenylalanine hydroxylase activities were found to be higher in cells adapted to a medium containing L-phenylalanine or L-tyrosine as the sole carbon source than in those grown in L-asparagine. The substrate of the reductase is quinonoid dihydropteridine, and the product is tentatively identified as a tetrahydropteridine through its ability to serve as a cofactor for phenylalanine hydroxylase. The enzyme shows no marked specificity for the pteridine cofactor that occurs naturally in this organism, L-threo-neopterin. The pH optimum for the reductase is 7.2, and nicotinamide adenine dinucleotide, reduced form, is the preferred cosubstrate. Inhibition of the reduced and untreated enzyme by several sulfhydryl reagents was observed. A metal requirement for the reductase could not be demonstrated. Dihydropteridine reductase was found to be inhibited by aminopterin in a competitive manner with respect to the quinonoid dihydro form of 2-amino-4-hydroxy-6,7-dimethyl-5,6,7,8-tetrahydropteridine.     

Dihydropteridine reductase from bovine liver. Purification, crystallization, and isolation of a binary complex with NADH.

Dihydropteridine reductase [EC 1.6.99.7] was purified from bovine liver in 50% yield and crystallized. The physicochemical properties of the purified enzyme were quite similar to those of sheep liver dihydropteridine reductase. During the course of purification, however, the enzyme was found to be separated into 2 major peaks together with minor peaks by column chromatography on CM-Sephadex, and one of the major peaks was identified as a binary complex of the enzyme with NADH. The reductase-NADH complex was also prepared in vitro and crystallized. Upon addition of quinonoid-dihydropterin to the complex, NADH was oxidized and released from the enzyme. The amount of bound NADH was calculated to be 2 moles per mole of the reductase. The occurrence of the reductase-NADH was calculated to be 2 moles per mole of the reductase. The occurrence of the reductase-NADH complex in bovine liver extract as a predominant form was in accord with the pyridine nucleotide specificity for NADH as a coenzyme. The results further support the view that NADH is the natural coenzyme of this reductase.     

Isolation and characterization of dihydropteridine reductase from human liver.

Dihydropteridine reductase (EC 1.6.99.7) was purified from human liver obtained at autopsy by a three-step chromatographic procedure with the use of (1) a naphthoquinone affinity adsorbent, (2) DEAE-Sephadex and (3) CM-Sephadex. The enzyme was typically purified 1000-fold with a yield of 25%. It gave a single band on non-denaturing and sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, and showed one spot on two-dimensional gel electrophoresis. The molecular weight of the enzyme was determined to be 50000 by sedimentation-equilibrium analysis and 47500 by gel filtration. On sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, a single subunit with mol.wt. 26000 was observed. A complex of dihydropteridine reductase with NADH was observed on gel electrophoresis. The isoelectric point of the enzyme was estimated to be pH 7.0. Amino acid analysis showed a residue composition similar to that seen for the sheep and bovine liver enzymes. The enzyme showed anomalous migration in polyacrylamide-gel electrophoresis. A Ferguson plot indicated that this behaviour is due to a low net charge/size ratio of the enzyme under the electrophoretic conditions used. The kinetic properties of the enzyme with tetrahydrobiopterin, 2-amino-4-hydroxy-6,7-dimethyl-5,6,7,8-tetrahydropteridine, NADH and NADPH are compared, and the effects of pH, temperature and a number of different compounds on catalytic activity are presented.     

Role of aspartate-37 in determining cofactor specificity and binding in rat liver dihydropteridine reductase

Full-length rat dihydropteridine reductase (DHPR) cDNAs have been combined with a prokaryotic expression vector and introduced into Escherichia coli. Transformed bacteria express dihydropteridine reductase immunoreactive proteins and demonstrate conversion of quinonoid dihydropteridines to their tetrahydro forms. Several recombinant enzymes have been purified to homogeneity and biochemical studies have been carried out comparing their properties with those exhibited by the rat liver enzyme. The optimal reaction conditions, kinetic constants, and stability are similar for the recombinant and naturally occurring enzyme. The results indicate that the nonmutant recombinant rat DHPR is an authentic replica of the natural protein and that the characteristics of DHPR activity are determined by a single gene product and do not require specific modification via the eukaryotic cell. In addition to the wild type, three specific mutagenic forms of the reductase, A-6-V, W-104-F, and D-37-I, and an additional abbreviated structure have also been formed. Each of the products exhibits reductase activity, although they show varied affinities for their cofactor, NADH, and less stability to chromatography, dialysis, and concentration than the wild-type enzyme. The N-terminal sequence contains a classic NADH binding region between amino acids 9 and 36, and Asp 37 is essential for binding the cofactor as is shown by the approximately 20-fold increase in dissociation constant for the D-37-I mutant and diminished kcat (approximately 43 s-1 compared to 156 s-1 for the wild-type enzyme). The results indicate that the DHPR cofactor binding site is similar to typical dinucleotide requiring dehydrogenases such as lactic acid and liver alcohol dehydrogenase.     

Production of antibodies to sheep liver dihydropteridine reductase: Characterization and use to study the enzyme defect in a variant form of phenylketonuria

An antiserum to sheep liver dihydropteridine reductase has been prepared in rabbits. The antiserum cross-reacts with dihydropteridine reductases from human, rat and bovine tissues. Using this antiserum, it was not possible to detect any cross-reacting material in the liver of a phenylketonuric child whose genetic defect has been shown to be due to a lack of detectable dihydropteridine reductase activity.     

Mevalonolactone inhibits the rate of synthesis and enhances the rate of degradation of 3-hydroxy-3-methylglutaryl coenzyme A reductase in rat hepatocytes

3-Hydroxy-3-methylglutaryl(HMG)-coenzyme A reductase purified from rat liver in the absence of protease inhibitors is composed of two distinct polypeptides of Mr = 51,000 and 52,500. Antibody raised to enzyme purified from rats fed a diet supplemented with cholestyramine and mevinolin inactivated HMG-CoA reductase. The antibody specifically precipitated a polypeptide of Mr = 94,000 from rat liver cells that had been previously incubated with [35S]methionine. The immunoprecipitation of the 35S-labeled polypeptide of Mr = 94,000 was prevented by addition of unlabeled pure HMG-CoA reductase (Mr = 51,000 and 52,500). Incubation of rat liver cells with mevalonolactone resulted in a decreased activity of HMG-CoA reductase and in a 40% decrease in the rate of incorporation of [35S]methionine into the immunoprecipitable reductase polypeptide of Mr = 94,000. In pulse-chase experiments, mevalonolactone enhanced the rate of degradation of the Mr = 94,000 polypeptide 3-fold. We propose that endogenous microsomal HMG-CoA reductase has a subunit of Mr = 94,000 and that the synthesis and degradation of this polypeptide are regulated by either mevalonolactone or, more likely, a product of mevalonolactone metabolism.     

Determination of NADPH-specific dihydropteridine reductase in extract from human, monkey, and bovine livers by single radial immunodiffusion: selective assay differentiating NADPH- and NADH-specific enzymes

It has been difficult to determine exactly NADPH-specific dihydropteridine reductase [EC 1.6.99.10] in samples which also contain NADH-specific dihydropteridine reductase [EC 1.6.99.7], because the latter enzyme interferes with the activity measurement of the former. We have devised a method to measure selectively the NADPH-specific reductase in crude extracts of bovine, human and monkey livers by the single radial immunodiffusion method using specific antiserum against the enzyme. This method makes it possible to determine the enzyme amount in 5 microliters of the 3-volume extracts of the livers. The amounts of NADPH-specific dihydropteridine reductase were calculated to be 0.252, 0.296, and 0.583 munits/5 microliter of the extracts of bovine, human, and monkey livers, respectively.     

Pteridine cofactor of phenylalanine hydroxylase in fetal rat liver

1. Pteridine cofactor of phenylalanine hydroxylase (EC 1.14.16.1) and dihydropteridine reductase (EC 1.6.99.7) in the phenylalanine hydroxylating system have been studied in the fetal rat liver. 2. Activities of pteridine cofactor and dihydropteridine reductase were measured as about 6 and 50%, respectively, of the levels of adult liver in the liver from fetuses on 20 days of gestation, at this stage the activity of phenylalanine hydroxylase was almost negligible in the liver. 3. Development of the activity of sepiapterin reductase (EC 1.1.1.153), an enzyme involved in the biosynthesis of pteridine cofactor, was studied in rat liver during fetal (20-22 days of gestation), neonatal and adult stages comparing with the activity of dihydrofolate reductase (EC 1.5.1.3). Activities of the enzymes were about 80 and 50%, respectively, of the adult levels at 20 days of gestation. 4. Some characteristics of sepiapterin reductase and dihydropteridine reductase of fetal liver were reported.     

Rat Striatal Synaptosomes as a Model System for Studying the Inhibition of Dihydropteridine Reductase Activity

Abstract

The distribution of dihydropteridine reductase between soluble and particulate fractions in synaptosomes parallels that of lactate dehydrogenase, but not monoamine oxidase. K_i and I₅₀ values for inhibitors obtained with the enzyme-rich P₂ fraction and its twice-washed fraction (P₂ W₂) were essentially the same, and were similar to those obtained with highly purified human liver enzyme. Dihydropteridine reductase inhibitory potency of multi-ring compounds containing a catechol-moiety was greater than that of single ring catecholic compounds, which in turn was greater than that of phydroxyphenolic compounds. The P₂ fraction of rat striatal synaptosomal preparations may serve as a convenient source of dihydropteridine reductase for studying the inhibition of this enzyme.