The NprA nitroreductase required for 2,4-dinitrophenol reduction in Rhodobacter capsulatus is a dihydropteridine reductase.

The Rhodobacter capsulatus nprA gene codes for a putative nitroreductase. A recombinant His(6)-NprA protein was overproduced in Escherichia coli and purified by affinity chromatography. This protein contained FMN and showed nitroreductase activity with a wide range of nitroaromatic compounds, such as 2-nitrophenol, 2,4-dinitrophenol, 2,6-dinitrophenol, 2,4,6-trinitrophenol (picric acid), 2,4-dinitrobenzoate and 2,4-dinitrotoluene, and with the nitrofuran derivatives nitrofurazone and furazolidone. NADPH was the main electron donor and the ortho nitro group was preferably reduced to the corresponding amino derivative. The apparent K(m) values of NprA for NADPH, 2,4-dinitrophenol, picric acid and furazolidone were 40 microM, 78 microM, 72 microM and 83 microM, respectively, at pH and temperature optima (pH 6.5, 30 degrees C). Escherichia coli cells overproducing the NprA protein were much more sensitive to the prodrug 5-(aziridin-1-yl)-2,4-dinitrobenzamide (CB1954) used in cancer therapy than non-transformed cells. NprA showed the highest activity with the quinonoid form of 6,7-dimethyl-7,8-dihydropterine as substrate, so that NprA may be involved in the synthesis of tetrahydrobiopterin in R. capsulatus. Expression of a transcriptional nprA-lacZ gene fusion was induced by phenylalanine or tyrosine, but not by other amino acids like glutamate or alanine. Furthermore, both nitroreductase activity and phenylalanine assimilation were inhibited in vivo by ammonium. A mutant defective in the nprA gene showed better growth rate with Phe or Tyr as nitrogen source than the wild-type strain, although both strains showed similar growth in media with Glu or without added nitrogen. These results suggest that the NprA nitroreductase may act in vivo as a dihydropteridine reductase involved in aromatic amino acids metabolism.

A survey of methods for the purification of microbial flavohemoglobins.

Over the past decade, the flavohemoglobin Hmp has emerged as the most significant nitric oxide (NO)-detoxifying protein in many diverse organisms, including yeasts and fungi but particularly pathogenic bacteria. Flavohemoglobins--the best-characterized class of microbial globin--comprise two domains: a globin domain with a noncovalently bound heme B and a flavin domain with recognizable binding sites for FAD and NAD(P)H. Hmp was first identified in Escherichia coli and now has a clearly defined role in NO biology in that organism: its synthesis is markedly up-regulated by NO, and hmp knockout mutants of E. coli and Salmonella typhimurium are severely compromised for survival in the presence of NO in vitro and in pathogenic lifestyles. In the presence of molecular O2, Hmp catalyzes an oxygenase or denitrosylase reaction in which NO is stoichiometrically converted to nitrate ion, which is relatively innocuous. In this chapter, we present a survey of the methods used to express and purify the flavohemoglobins from diverse microorganisms and describe in more detail three methods developed and used in this laboratory for the E. coli protein. Particular problems are highlighted, particularly (a) the toxic consequences of Hmp overexpression that result from its ability to catalyze partial oxygen reduction and (b) the expression of protein with substoichiometric content of redox-active flavin and heme centers.

Crystallization and preliminary characterization of dihydropteridine reductase from Dictyostelium discoideum.

Dihydropteridine reductase from Dictyostelium discoideum (dicDHPR) can produce D-threo-BH(4) [6R-(1'R,2'R)-5,6,7,8-tetrahydrobiopterin], a stereoisomer of L-erythro-BH(4), in the last step of tetrahydrobiopterin (BH(4)) recycling. In this reaction, DHPR uses NADH as a cofactor to reduce quinonoid dihydrobiopterin back to BH(4). To date, the enzyme has been purified to homogeneity from many sources. In this report, the dicDHPR-NAD complex has been crystallized using the hanging-drop vapour-diffusion method with PEG 3350 as a precipitant. Rectangular-shaped crystals were obtained. Crystals grew to maximum dimensions of 0.4 x 0.6 x 0.1 mm. The crystal belonged to space group P2(1), with unit-cell parameters a = 49.81, b = 129.90, c = 78.76 A, beta = 100.00 degrees , and contained four molecules in the asymmetric unit, forming two closely interacting dicDHPR-NAD dimers. Diffraction data were collected to 2.16 A resolution using synchrotron radiation. The crystal structure has been determined using the molecular-replacement method.

Expression and Purification of Quinine Dihydro Pteridine Reductase from astrocytes and its significance in the astrocyte pathology.

Quinine dihydropteridinereductase (QDPR) is involved in the synthesis of tetradihydrobiopteridine (BH4) that serve as cofactor for many aromatic hydroxylases including induced nitric oxide synthase (NOS) leading to NO production. Increased activity of QDPR has been associated with decrease levels of TGF-ß, a cytokine that regulates the immune response and that elevated levels of NO has been associated with neurodegenerative diseases. Thus, expression of QDPR in astrocytes is essential to study the pathological changes observed in many neurodegenerative disorders. We have expressed QDPR in astrocytes and generated stably expressing clones that overexpresses QDPR. We further verified the specificity of QDPR expression using immunofluorescence and immunoblotting. To further confirm, we purified QDPR using Ni-NTA column and subjected the purified fraction to immunoblotting using anti-QDPR antibody and identified two major protein products of QDPR resolving at 25 and 17 kDa as reported in the literature. In order to further assess the significance of QDPR expression, we verified the expression of iNOS in QDPR over expressing cells. We show for the first time statistically significant up regulation of iNOS in QDPR overexpressing astrocytes. Increased expression of iNOS associated with astrocyte pathology seen in many neurodegenerative disorders may have implications in autoimmune neurodegenerative disorders.

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.

A simple procedure for purification of NADH-specific dihydropteridine reductase from mammalian liver.

A simple and convenient purification method which can yield a homogeneous preparation from even a small amount of starting material was devised for NADH-specific dihydropteridine reductase from rat liver. The procedure is essentially composed of two steps, i.e., affinity chromatography on Matrex gel blue A and hydrophobic chromatography on Phenyl-Sepharose. Prior to the Matrex gel blue A chromatography, the crude extract of rat liver was oxidized to dissociate NADH, bound in the enzyme-NADH complex, from the enzyme. Low molecular weight substances in the extract were removed by Sephadex G-25 gel filtration; then the enzyme was purified by successive chromatographies on Matrex gel blue A and Phenyl-Sepharose columns. Thus about 0.1 mg of purified enzyme was obtained from 3 g of rat liver with 40% recovery. The preparation showed a single protein band on polyacrylamide and SDS-gel electrophoresis. Using NADH and tetrahydro-6-methylpterin, the maximal velocity of the enzyme was determined to be 64.2 mumol quinonoid-dihydro-6-methylpterin reduced/min/mg. Km values of the enzyme were 0.85 microM and 3.4 microM for NADH and quinonoid-dihydro-6-methylpterin, respectively. This simple purification method was also applicable to livers from other mammalian sources such as human and bovine.

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.

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.

Identification of dihydropteridine reductase in human platelets.

Normal human platelets were shown to contain the enzyme dihydropteridine reductase. The enzyme was not found in a variety of other cells of hematogenous origin. Partial purification and kinetic and physical data indicated that the platelet enzyme is similar to that previously characterized from liver. Dihydropteridine reductase is important for the regeneration of tetrahydrobiopterin, a required cofactor in hydroxylation reactions involved in biogenic amine formation. The presence of the enzyme may indicate that some synthesis de novo of serotonin and/or catecholamines occurs in platelets, as opposed to a purely storage and transport function. In addition, screening for hyperphenylalaninemia due to dihydropteridine reductase deficiency may become feasible by assaying platelets for enzyme activity.

Purification of dihydropteridine reductase from human platelets.

Dihydropteridine reductase was purified approximately 1,700-fold from human outdated blood platelets. Two forms of the enzyme, A and B, were resolved. They have the same Km values for 2-amino-6,7,-dimethyl-4-hydroxydihydropteridine (46 microM vs 49 microM), but the A form has a Km for NADH that is two times higher than that of the B form (20 microM vs 9 microM).

Purification of dihydropterin reductase using immobilized Cibacron Blue.

Chromatography on columns of immobilized Cibacron Blue (Blue Dextran--agarose) can be used as a major step in the purification of quinonoid dihydropterin reductase. The reductase has been isolated from fractions of beef kidney by selective binding to the immobilized Cibacron in the presence of tetrahydropterin. The binding of the reductase to Blue Dextran and its specific elution from columns of Blue Dextran--agarose indicate that the reductase possesses the dinucleotide (NAD+) binding domain. The results of kinetic experiments give validity to both our affinity chromatography of the reductase and to an ordered mechanism for the formation of tetrahydropterin. Chromatography on Blue Dextran--agarose has been used to show that folate or amethopterin can compete with Cibacron Blue for the dinucleotide domain of the reductase. The p-aminobenzoyl-glutamate moiety of the folates competes with Cibacron Blue for the NADH site of the reductase. A stable binary complex of dihydropterin reductase with NADH has been detected by gel electrophoresis.

A naphthoquinone adsorbent for affinity chromatography of human dihydropteridine reductase.

1. A 1,2-naphthoquinone adsorbent is described which allows simple purification of dihydropteridine reductase directly from crude extract. 2. The native molecular weight indicates that a tetramer structure of the species is isolated by this method; this is unusual and possibly reflects the capacity of the procedure to preserve the native state of the enzyme.

Dihydropteridine reductase from Escherichia coli exhibits dihydrofolate reductase activity.

E. coli Dihydropteridine reductase, known to have a pterin-independent oxidoreductase activity with potassium ferricyanide as electron donor, has now been shown to possess also dihydrofolate reductase activity. The kinetic parameters for dihydrofolate reductase activity have been determined. The ratio of the three activities, dihydropteridine reductase, dihydrofolate reductase and pterin-independent oxidoreductase activity is 1.0, 0.05 and 4.3, respectively. The enzyme, a flavoprotein which is unstable in the presence of dithiothreitol, was shown to be a monomer with a molecular mass of 25.7 kDa. The apparent lack of discrimination between hydride transfer from the pyridine nucleotide to N5 of the pterin in the dihydropteridine reductase reaction and C6 of folate in the dihydrofolate reaction suggested that the FAD prosthetic group may be involved in the hydride transfers. The flavoprotein inhibitor N,N- dimethylpropargylamine inhibited the dihydropteridine reductase and oxidoreductase reactions differently and did not affect the dihydrofolate reductase activity however.