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.     

Residues of E. coli topoisomerase I conserved for interaction with a specific cytosine base to facilitate DNA cleavage

Bacterial and archaeal topoisomerase I display selectivity for a cytosine base 4 nt upstream from the DNA cleavage site. Recently, the solved crystal structure of Escherichia coli topoisomerase I covalently linked to a single-stranded oligonucleotide revealed that R169 and R173 interact with the cytosine base at the −4 position via hydrogen bonds while the phenol ring of Y177 wedges between the bases at the −4 and the −5 position. Substituting R169 to alanine changed the selectivity of the enzyme for the base at the −4 position from a cytosine to an adenine. The R173A mutant displayed similar sequence selectivity as the wild-type enzyme, but weaker cleavage and relaxation activity. Mutation of Y177 to serine or alanine rendered the enzyme inactive. Although mutation of each of these residues led to different outcomes, R169, R173 and Y177 work together to interact with a cytosine base at the −4 position to facilitate DNA cleavage. These strictly conserved residues might act after initial substrate binding as a Molecular Ruler to form a protein–DNA complex with the scissile phosphate positioned at the active site for optimal DNA cleavage by the tyrosine hydroxyl nucleophile to facilitate DNA cleavage in the reaction pathway.     

DIHYDROPTERIDINE REDUCTASE MAY FUNCTION IN TETRAHYDROFOLATE METABOLISM

Abstract— The tetrahydrofolate-dependent serine hydroxymethyl transferase ( l -serine: tetrahydrofolate 10-hydroxymethyl transferase, EC 2.1.2.1) reaction in rat or human brain homogenates incubated aerobically is dependent on added reducing agents for full activity in order to protect the readily oxidized substrate, tetrahydrofolate. In this role, 0.1 m m -NADH is as affective as 10m m -2-mercaptoethanol and it can be shown that the NADH prevents destruction of tetrahydrofolate incubated with brain homogenates. If the dihydropteridine reductase (NADPH:6,7-dihydropteridine oxidoreductase, EC 1.6.99.7) activity of the brain homogenate is inhibited by a specific antiserum, NADH, but not 2-mercaptoeth-anol, is no longer effective. Furthermore, an homogenate of a brain biopsy from a human lacking dihydropteridine reductase requires added dihydropteridine reductase for maximal stimulation by NADH of the serine hydroxymethyl transferase reaction. We conclude that dihydropteridine reductase mediates the NADH stimulation and can play a role in preserving tetrahydrofolate from oxidation. The rinding of greatly reduced folate levels in the brain biopsy from the human lacking dihydropteridine reductase supports this postulated role of dihydropteridine reductase in folate metabolism.     

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.     

Switching base preferences of mismatch cleavage in endonuclease V: an improved method for scanning point mutations

Endonuclease V (endo V) recognizes a broad range of aberrations in DNA such as deaminated bases or mismatches. It nicks DNA at the second phosphodiester bond 3′ to a deaminated base or a mismatch. Endonuclease V obtained from Thermotoga maritima preferentially cleaves purine mismatches in certain sequence context. Endonuclease V has been combined with a high-fidelity DNA ligase to develop an enzymatic method for mutation scanning. A biochemical screening of site-directed mutants identified mutants in motifs III and IV that altered the base preferences in mismatch cleavage. Most profoundly, a single alanine substitution at Y80 position switched the enzyme to essentially a C-specific mismatch endonuclease, which recognized and cleaved A/C, C/A, T/C, C/T and even the previously refractory C/C mismatches. Y80A can also detect the G13D mutation in K-ras oncogene, an A/C mismatch embedded in a G/C rich sequence context that was previously inaccessible using the wild-type endo V. This investigation offers insights on base recognition and active site organization. Protein engineering in endo V may translate into better tools in mutation recognition and cancer mutation scanning.     

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.     

Localization of the human dihydropteridine reductase gene to band p15.3 of chromosome 4 by in situ hybridization

We report the localization of the gene for dihydropteridine reductase (DHPR) to the human chromosome region 4p15.3 by in situ hybridization using a cDNA probe to the enzyme. The distal end of the short arm of chromosome 4 is of considerable interest because the gene responsible for Huntington's disease is located in this region. Although this part of the chromosome is being extensively studied, DHPR is the first well-characterised gene to be assigned to the region. Restriction enzyme fragment length polymorphisms have been detected with a number of restriction endonucleases, including AvaII and MspI. These features may make the DHPR cDNA clone a useful probe not only for prenatal diagnosis of DHPR deficiency but also for linkage studies of Huntington's disease.     

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.     

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.     

Dihydropteridine reductase as an alternative to dihydrofolate reductase for synthesis of tetrahydrofolate in Thermus thermophilus

A strategy devised to isolate a gene coding for a dihydrofolate reductase from Thermus thermophilus DNA delivered only clones harboring instead a gene (the T. thermophilus dehydrogenase [DH(Tt)] gene) coding for a dihydropteridine reductase which displays considerable dihydrofolate reductase activity (about 20% of the activity detected with 6,7-dimethyl-7,8-dihydropterine in the quinonoid form as a substrate). DH(Tt) appears to account for the synthesis of tetrahydrofolate in this bacterium, since a classical dihydrofolate reductase gene could not be found in the recently determined genome nucleotide sequence (A. Henne, personal communication). The derived amino acid sequence displays most of the highly conserved cofactor and active-site residues present in enzymes of the short-chain dehydrogenase/reductase family. The enzyme has no pteridine-independent oxidoreductase activity, in contrast to Escherichia coli dihydropteridine reductase, and thus appears more similar to mammalian dihydropteridine reductases, which do not contain a flavin prosthetic group. We suggest that bifunctional dihydropteridine reductases may be responsible for the synthesis of tetrahydrofolate in other bacteria, as well as archaea, that have been reported to lack a classical dihydrofolate reductase but for which possible substitutes have not yet been identified.     

Regulation of GTP cyclohydrolase I and dihydropteridine reductase in rat pheochromocytoma PC 12 cells

The addition of 8-bromo cyclic AMP, forskolin, theophylline, and 3-isobutyl-1-methylxanthine to the medium of PC 12 cells resulted in an increase in GTP cyclohydrolase I activity, but had no effect on dihydropteridine reductase activity, except theophylline which caused a decrease in dihydropteridine reductase activity at 96 h. GTP cyclohydrolase I activity peaked at 24 h and returned to normal 96 h after drug treatment. Cycloheximide decreased GTP cyclohydrolase I activity at 48 and 96 h, but had little effect on dihydropteridine reductase activity. The addition of reserpine selectively increased only GTP cyclohydrolase I activity. The addition of tetrahydrobiopterin and sepiapterin, however, coordinately inhibited both GTP cyclohydrolase I and dihydropteridine reductase activities. It appears that GTP cyclohydrolase I activity in PC 12 cells is regulated by cyclic AMP stimulation and by end-product inhibition, whereas dihydropteridine reductase activity is only subject to pterin inhibition.     

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.     

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.     

Spontaneous splicing mutations at the dihydrofolate reductase locus in Chinese hamster ovary cells.

We isolated and characterized three spontaneous mutants of Chinese hamster ovary cells that were deficient in dihydrofolate reductase activity. All three mutants contained no detectable enzyme activity and produced dihydrofolate reductase mRNA species that were shorter than those of the wild type by about 120 bases. Six exons are normally represented in this mRNA; exon 5 was missing in all three mutant mRNAs. Nuclease S1 analysis of the three mutants indicated that during the processing of the mutant RNA, exon 4 was spliced to exon 6. The three mutant genes were cloned, and the regions around exons 4 and 5 were sequenced. In one mutant, the GT dinucleotide at the 5' end of intron 5 had changed to CT. In a second mutant, the first base in exon 5 had changed from G to T. In a revertant of this mutant, this base was further mutated to A, a return to a purine. Approximately 25% of the mRNA molecules in the revertant were spliced correctly to produce an enzyme with one presumed amino acid change. In the third mutant, the AG at the 3' end of intron 4 had changed to AA. A mutation that partially reversed the mutant phenotype had changed the dinucleotide at the 5' end of intron 4 from GT to AT. The splicing pattern in this revertant was consistent with the use of cryptic donor and acceptor splice sites close to the original sites to produce an mRNA with three base changes and a protein with two amino acid changes. These mutations argue against a scanning model for the selection of splice site pairs and suggest that only a single splice site need be inactivated to bring about efficient exon skipping (a regulatory mechanism for some genes). The fact that all three mutants analyzed exhibited exon 5 splicing mutations indicates that these splice sites are hot spots for spontaneous mutation.     

Identification of a physiological phosphorylation site of the herpes simplex virus 1-encoded protein kinase Us3 which regulates its optimal catalytic activity in vitro and influences its function in infected cells

Us3 is a serine/threonine protein kinase encoded by herpes simplex virus 1 (HSV-1). Here, we report the identification of a physiological Us3 phosphorylation site on serine at position 147 (Ser-147) which regulates its protein kinase activity in vitro. Moreover, mutation of this site influences Us3 function, including correct localization of the enzyme and induction of the usual morphological changes in HSV-1-infected cells. These conclusions are based on the following observations: (i) in in vitro kinase assays, a domain of Us3 containing Ser-147 was specifically phosphorylated by Us3 and protein kinase A, while a mutant domain in which Ser-147 was replaced with alanine was not; (ii) in vitro, alanine replacement of Ser-147 (S147A) in Us3 resulted in significant impairment of the kinase activity of the purified molecule expressed in a baculovirus system; (iii) phosphorylation of Ser-147 in Us3 tagged with the monomeric fluorescent protein (FP) VenusA206K (VenusA206K-Us3) from Vero cells infected with a recombinant HSV-1 encoding VenusA206K-Us3 was specifically detected using an antibody that recognizes phosphorylated serine or threonine residues with arginine at the -3 and -2 positions; and (iv) the S147A mutation influenced some but not all Us3 functions, including the ability of the protein to localize itself properly and to induce wild-type cytopathic effects in infected cells. Our results suggest that some of the regulatory activities of Us3 in infected cells are controlled by phosphorylation at Ser-147.     

SNPs detection by a single-strand specific nuclease on a PNA zip-code microarray

In this report, a reliable peptide nucleic acid (PNA) microarray-based method for accurately detecting single nucleotide polymorphism (SNP) in human genes is described. The technique relies on the mismatched cleavage activity of a single-strand specific (SSS) nuclease. PCR amplification was performed to prepare gene fragments containing the mutation sites. The amplified fragments were then employed as templates for the SSS nuclease reaction using chimeric probes, modified with biotin at the 5' end and extended with a unique anchoring zip-code complement sequence at the 3' end. The SSS nuclease promotes cleavage of heteroduplex DNAs at base mismatched positions to produce crumbled chimeric probes in the presence of imperfectly matching template strands. In contrast, the probes remain intact when they interact with perfectly matched template strands. Only the non-fragmented probes generate fluorescence signals after treatment with streptavidin-Cy3 on the PNA zip-code array. This methodology was used to successfully genotype selected Korean-specific BRCA mutation sites with wild type and mutant samples. The investigation has led to the development of a reliable SSS nuclease-based system for the diagnosis of human genetic mutations or SNPs.     

Preliminary studies on the primary structure of rat liver dihydropteridine reductase

Cyanogen bromide cleavage of reductively alkylated homogeneous rat liver dihydropteridine reductase afforded several peptide fragments identifiable by polyacrylamide electrophoresis of which 6 (CB-1 to CB-6) could be individually isolated by C8 reverse phase HPLC. Each was characterised by N-terminal amino acid analysis and sequence information was derived for CB-1, CB-4 and CB-6. The blocked N-terminal of the holoenzyme was identified as pyroglutamate and the C-terminal sequence was obtained by sequential degradation.     

Molecular and immunological comparison of human dihydropteridine reductase in liver, cultured fibroblasts and continuous lymphoid cells.

An antiserum was raised in a rabbit against highly purified human liver dihydropteridine reductase (EC 1.6.99.7). Dihydropteridine reductase from human liver, in human cultured fibroblasts and in continuous lymphoid cells all showed identical antigenic properties. The structural characteristics of the reductase from these three sources were further compared by the use of high-precision two-dimensional polyacrylamide-gel electrophoresis. The enzyme from radiolabelled fibroblasts and continuous lymphoid cells was isolated by immunoprecipitation or by affinity chromatography and compared with the purified liver enzyme. Two major polypeptide species were resolved, and polypeptides from all three sources co-migrated identically. Indirect evidence is presented indicating that one of the polypeptide species may have been derived from the other via a post-translational modification. These results support the concept that the same structural gene(s) encodes for dihydropteridine reductase in human liver, fibroblasts and lymphocytes.     

Base-flipping dynamics in a DNA hairpin processing reaction

Many enzymes that repair or modify bases in double-stranded DNA gain access to their substrates by base flipping. Although crystal structures provide stunning snap shots, biochemical approaches addressing the dynamics have proven difficult, particularly in complicated multi-step reactions. Here, we use protein–DNA crosslinking and potassium permanganate reactivity to explore the base-flipping step in Tn5 transposition. We present a model to suggest that base flipping is driven by a combination of factors including DNA bending and the intrusion of a probe residue. The forces are postulated to act early in the reaction to create a state of tension, relieved by base flipping after cleavage of the first strand of DNA at the transposon end. Elimination of the probe residue retards the kinetics of nicking and reduces base flipping by 50%. Unexpectedly, the probe residue is even more important during the hairpin resolution step. Overall, base flipping is pivotal to the hairpin processing reaction because it performs two opposite but closely related functions. On one hand it disrupts the double helix, providing the necessary strand separation and steric freedom. While on the other, transposase appears to position the second DNA strand in the active site for cleavage using the flipped base as a handle.     

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.