Synthesis of biopterin from dihydroneopterin triphosphate by rat tissues

High performance liquid chromatography procedure for the analysis of pterins of biopterin synthesis from dihydroneopterin triphosphate via sepiapterin in rat tissues has been described. Sepiapterin-synthesizing enzyme 1, which catalyzes in the presence of Mg²⁺ the conversion of dihydroneopterin triphosphate to an intermediate designated compound X was assayed by determining pterin which is formed from compound X under acidic conditions. Sepiapterin- and biopterin-synthesizing activity were also assayed by determining sepiapterin and biopterin, respectively. Analytical results revealed the presence of these activities in most rat tissues examined and high levels were found in kidney, pineal gland and liver. Activities were also detectable in peripheral erythrocytes.     

Structure and function of the E. coli dihydroneopterin triphosphate pyrophosphatase: a Nudix enzyme involved in folate biosynthesis

Nudix hydrolases are a superfamily of pyrophosphatases, most of which are involved in clearing the cell of potentially deleterious metabolites and in preventing the accumulation of metabolic intermediates. We determined that the product of the orf17 gene of Escherichia coli, a Nudix NTP hydrolase, catalyzes the hydrolytic release of pyrophosphate from dihydroneopterin triphosphate, the committed step of folate synthesis in bacteria. That this dihydroneopterin hydrolase (DHNTPase) is indeed a key enzyme in the folate pathway was confirmed in vivo: knockout of this gene in E. coli leads to a marked reduction in folate synthesis that is completely restored by a plasmid carrying the gene. We also determined the crystal structure of this enzyme using data to 1.8 A resolution and studied the kinetics of the reaction. These results provide insight into the structural bases for catalysis and substrate specificity in this enzyme and allow the definition of the dihydroneopterin triphosphate pyrophosphatase family of Nudix enzymes.     

Histidine 179 mutants of GTP cyclohydrolase I catalyze the formation of 2-amino-5-formylamino-6-ribofuranosylamino-4(3H)-pyrimidinone triphosphate.

GTP cyclohydrolase I catalyzes the conversion of GTP to dihydroneopterin triphosphate. The replacement of histidine 179 by other amino acids affords mutant enzymes that do not catalyze the formation of dihydroneopterin triphosphate. However, some of these mutant proteins catalyze the conversion of GTP to 2-amino-5-formylamino-6-ribofuranosylamino-4(3H)-pyrimidinone 5'-triphosphate as shown by multinuclear NMR analysis. The equilibrium constant for the reversible conversion of GTP to the ring-opened derivative is approximately 0.1. The wild-type enzyme converts the formylamino pyrimidine derivative to dihydroneopterin triphosphate; the rate is similar to that observed with GTP as substrate. The data support the conclusion that the formylamino pyrimidine derivative is an intermediate in the overall reaction catalyzed by GTP cyclohydrolase I.     

Purification and properties of the enzymes from Drosophila melanogaster that catalyze the synthesis of sepiapterin from Dihydroneopterin triphosphate

Sepiapterin synthase, the enzyme system responsible for the synthesis of sepiapterin from dihydroneopterin triphosphate, has been partially purified from extracts of the heads of young adult fruit flies (Drosophila melanogaster). The sepiapterin synthase system consists of two components, termed enzyme A (MW 82,000) and enzyme B (MW 36,000). Some of the properties of the enzyme system are as follows: NADPH and a divalent cation, supplied most effectively as MgCl₂, are required for activity; optimal activity occurs at pH 7.4 and 30 C; the K _m for dihydroneopterin triphosphate is 10 µm; and a number of unconjugated pterins, including biopterin and sepiapterin, are inhibitory. Dihydroneopterin cannot be used as substrate in place of dihydroneopterin triphosphate. Evidence is presented in support of a proposed reaction mechanism for the enzymatic conversion of dihydroneopterin triphosphate to sepiapterin in which enzyme A catalyzes the production of a labile intermediate by nonhydrolytic elimination of the phosphates of dihydroneopterin triphosphate, and enzyme B catalyzes the conversion of this intermediate, in the presence of NADPH, to sepiapterin. An analysis of the activity of sepiapterin synthase during development in Drosophila revealed the presence of a small amount of activity in eggs and young larvae and a much larger amount in late pupae and young adults. Sepiapterin synthase activity during development corresponds with the appearance of sepiapterin in the flies. Of a variety of eye color mutants of Drosophila melanogaster tested for sepiapterin synthase activity, only purple (pr) flies contained activity that was significantly lower than that found in the wild-type flies (22% of the wild-type activity). Further studies indicated that the amount of enzyme A activity is low in purple flies, whereas the amount of enzyme B activity is equal to that present in wild-type flies.This work was supported by research grants from the National Institutes of Health (AM03442) and the National Science Foundation (PCM75-19513 A02). G. G. K. was supported as a predoctoral trainee by National Institutes of Health Training Grant GM00515.     

Dihydroneopterin triphosphate epimerase of Escherichia coli: purification, genetic cloning, and expression.

The enzyme catalyzing the epimerization at position 2' of dihydroneopterin triphosphate was purified by a factor of about 10,000 from cell extract of Escherichia coli. The cognate gene was cloned, sequenced, expressed, and mapped to kb 2427 on the E. coli chromosome.     

Biosynthesis of tetrahydrobiopterin: conversion of dihydroneopterin triphosphate to tetrahydropterin intermediates

It is known that the first step in the de novo synthesis of tetrahydrobiopterin from GTP is the conversion of GTP to dihydroneopterin triphosphate. Recent evidence supports the conclusion that beyond this first step, the pterin intermediates in the pathway are all at the tetrahydro level of reduction. We have now shown that partially purified fractions from rat liver, rat brain and bovine adrenal medulla catalyze the conversion of dihydroneopterin triphosphate to tetrahydrobiopterin, as well as to the putative intermediates in the pathway, 6-pyruvoyl-tetrahydropterin and 6-lactoyl-tetrahydropterin. Results of both enzymatic and chemical studies support the assigned structures for the latter two tetrahydropterins. We have also purified extensively from brain an enzyme, distinct from sepiapterin reductase, that catalyzes the TPNH-dependent reduction of 6-pyruvoyl-tetrahydropterin to 6-lactoyl-tetrahydropterin. The role of this reductase in tetrahydrobiopterin synthesis has not yet been established.     

Guanosine triphosphate cyclohydrolase activity in rat tissues.

The GTP cyclohydrolase activity of rat tissues has been studied by means of the measurement of formic acid release and neopterin synthesis from GTP. After gel filtration of a 45%-satd.-(NH4)2SO4 fraction of liver homogenates, three enzyme fractions were separated and named A1, A2 and A3 according to the order of their elution. Fractions A1 and A3 displayed an 8-formyl-GTP deformylase activity; no proof of cyclized product has yet been established. This activity was heat-labile and required Mg2+ for maximal activity. Fraction A2 displayed a 'neopterin-synthetase' activity, with dihydroneopterin triphosphate and formic acid formed in stochiometric amounts. Fraction A1 isolated from heat-treated homogenates also produced dihydroneopterin triphosphate. Neopterin synthetase activity in fractions A1 and A2 was heat-resistant and inhibited by Mg2+. In liver the A2 fraction represented 70-75% of the neopterin synthetase capacity and was inhibited by reduced pterines (sepiapterin, dihydrobiopterin and tetrahydrobiopterin) and to a lesser extent by reduced forms of folic acid. In kidney and brain, fraction A1 and A3 GTP 8-formylhydrolase activities were found in significant amounts, in contrast with the neopterin synthetase activity, which was low and appeared to be confined to the A1 fraction.     

Crystal structure of 7,8-dihydroneopterin triphosphate epimerase.

BACKGROUND: Dihydroneopterin triphosphate (H2NTP) is the central substrate in the biosynthesis of folate and tetrahydrobiopterin. Folate serves as a cofactor in amino acid and purine biosynthesis and tetrahydrobiopterin is used as a cofactor in amino acid hydroxylation and nitric oxide synthesis. In bacteria, H2NTP enters the folate biosynthetic pathway after nonenzymatic dephosphorylation; in vertebrates, H2NTP is used to synthesize tetrahydrobiopterin. The dihydroneopterin triphosphate epimerase of Escherichia coli catalyzes the inversion of carbon 2' of H2NTP. RESULTS: The crystal structure of the homo-octameric protein has been solved by a combination of multiple isomorphous replacement, Patterson search techniques and cyclic averaging and has been refined to a crystallographic R factor of 18.8% at 2.9 A resolution. The enzyme is a torus-shaped, D4 symmetric homo-octamer with approximate dimensions of 65 x 65 A. Four epimerase monomers form an unusual 16-stranded antiparallel beta barrel by tight association between the N- and C-terminal beta strands of two adjacent subunits. Two tetramers associate in a head-to-head fashion to form the active enzyme complex. CONCLUSIONS: The folding topology, quaternary structure and amino acid sequence of epimerase is similar to that of the dihydroneopterin aldolase involved in the biosynthesis of the vitamin folic acid. The monomer fold of epimerase is also topologically similar to that of GTP cyclohydrolase I (GTP CH-1), 6-pyrovoyl tetrahydropterin synthase (PTPS) and uroate oxidase (UO). Despite a lack of significant sequence homology these proteins share a common subunit fold and oligomerize to form central beta barrel structures employing different cyclic symmetry elements, D4, D5, D3 and D2, respectively. Moreover, these enzymes have a topologically equivalent acceptor site for the 2-amino-4-oxo pyrimidine (2-oxo-4-oxo pyrimidine in uroate oxidase) moiety of their respective substrates.     

Formation of beta,gamma-methylene-7,8-dihydroneopterin 3'-triphosphate from beta,gamma-methyleneguanosine 5'-triphosphate by GTP cyclohydrolase I of Escherichia coli

GTP cyclohydrolase I of Escherichia coli converts [beta,gamma-methylene] GTP to a fluorescent product that is characterized as [beta,gamma-methylene]dihydroneopterin triphosphate. Interaction between the GTP analog and the enzyme gave a Ki of 3.0 microM, which may be compared to the Km of 0.1 microM for GTP. This new analog of dihydroneopterin triphosphate may, in turn, be converted to the same greenish-yellow pteridines (compounds X, X1, and X2) that are obtained from dihydroneopterin triphosphate. Because of its stability to phosphatase action, this analog may be useful for studies in pteridine metabolism.     

Intermediates in the enzymic synthesis of tetrahydrobiopterin in Drosophila melanogaster

9 partially purified enzyme (Enzyme A) from Drosophila melanogaster Aatalyzes the conversion of 7,8- dihydroneopterin triphosphate to a compound that, from its ultraviolet absorption spectrum and other characteristics, appears to be 6- pyruvoyl -tetrahydropterin. This product can be converted to 6-lactoyl-tetrahydropterin in the presence of another partially purified enzyme (Enzyme B) and NADPH, and to 5,6,7,8-tetrahydrobiopterin in the presence of a third enzyme preparation (biopterin synthase) and NADPH. The enzymically-produced 6-lactoyl-tetrahydropterin, when exposed to air, is oxidized nonenzymically to sepiapterin (6-lactoyl-7,8- dihydropterin ). The results indicate that although 6-lactoyl-tetrahydropterin can be converted enzymically to tetrahydrobiopterin, neither it nor sepiapterin is an obligate intermediate in the conversion of 7,8- dihydroneopterin triphosphate to tetrahydrobiopterin.     

The enzymatic conversion of dihydroneopterin triphosphate to tripolyphosphate and 6-pyruvoyl-tetrahydropterin, an intermediate in the biosynthesis of other pterins in Drosophila melanogaster

The enzyme, previously called "sepiapterin synthase A," has been purified by approximately 700-fold from the heads of Drosophila melanogaster. This enzyme catalyzes the Mg2+-dependent conversion of 2-amino-4-oxo-6-(D-erythro-1',2',3'-trihydroxypropyl)-7,8-dihydrop teridine triphosphate (dihydroneopterin triphosphate or H2-NTP) to two products, one of which we have identified as tripolyphosphate. The other product is a phosphate-free, unstable compound which is an intermediate in the biosynthesis of several other naturally occurring pterins in Drosophila. This product is stable enough under anaerobic conditions to allow it to be characterized as 6-pyruvoyl-5,6,7,8-tetrahydropterin (6-pyruvoyl-H4-pterin). The 3-carbon side chain was identified as a pyruvoyl group on the basis of the susceptibility of the enzymatic product to reduction with tritiated sodium borohydride and the determination of the amounts and the sites of incorporation of tritium resulting from this reduction. From these observations, we suggest that this enzyme be renamed "6-pyruvoyl-H4-pterin synthase."     

Purification and properties of the enzymes from Drosophila melanogaster that catalyze the conversion of dihydroneopterin triphosphate to the pyrimidodiazepine precursor of the drosopterins

The enzyme system responsible for the conversion of 2-amino-4-oxo-6-(D-erythro-1',2',3'-trihydroxypropyl)-7,8-dihyd roptridine triphosphate (dihydroneopterin triphosphate or H2-NTP) to 2-amino-4-oxo-6-acetyl-7,8-dihydro-3H,9H-pyrimido[4,5-b]-[1,4]diazepine (pyrimidodiazepine or PDA), a precursor to the red eye pigments, he drosopterins, has been purified from the heads of Drosophila melanogaster. The PDA-synthesizing system consists of two components, a heat-stable enzyme and a heat-labile enzyme. The heat-stable enzyme can be replaced by sepiapterin synthase A, a previously purified enzyme required for the Mg2+-dependent conversion of H2-NTP to an unstable compound that appears to be 6-pyruvoyltetrahydropterin (pyruvoyl-H4-pterin). The heat-labile enzyme, purified to near-homogeneity and termed PDA synthase (Mr = 48,000), catalyzes the conversion of pyruvoyl-H4-pterin to PDA in a reaction requiring the presence of reduced glutathione. Because PDA is two electrons more reduced than pyruvoyl-H4-pterin, the reducing power required for this transformation is probably supplied by glutathione. The PDA-synthesizing system requires the presence of another thiol-containing compound such as 2-mercaptoethanol when incubation conditions 2-mercaptoethanol is no longer required. Evidence is presented to indicate that the Drosophila eye color mutant, sepia, is missing PDA synthase.     

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 approximately 83,000 and consists of four identical subunits, each of which has a molecular weight of approximately 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.     

Dyspropterin, an intermediate formed from dihydroneopterin triphosphate in the biosynthetic pathway of tetrahydrobiopterin

The structure of dyspropterin, a new name given to an intermediate which is formed from dihydroneopterin triphosphate in the biosynthetic pathway of tetrahydrobiopterin, has been studied. Sepiapterin reductase (EC 1.1.1.153) was found to reduce dyspropterin to tetrahydrobiopterin in the presence of NADPH. Several lines of evidence showing the formation of tetrahydrobiopterin have been presented. Stoichiometric analysis revealed that there is a 1:2 relationship between the production of biopterin and the oxidation of NADPH during the reductase-catalyzed reduction of dyspropterin. The tetrahydrobiopterin production from dyspropterin was enhanced by dihydropteridine reductase (EC 1.6.99.7). Dyspropterin could also serve as a cofactor in phenylalanine hydroxylase (EC 1.14.16.1) system. These results are consistent with the view that dyspropterin is 6-(1,2-dioxopropyl)-5,6,7,8-tetrahydropterin. Based on our findings, the biosynthetic pathway of tetrahydrobiopterin from dihydroneopterin triphosphate has been discussed.     

Lithosperm inhibition of anterior pituitary hormones.

The enzyme system for the synthesis of the pteridine pigment, sepiapterin, from $\text{2-amino-4-hydroxy-6-(D-}\text{erythro}\text{-1',2',3'-trihydroxyprophyl)}$ triphosphate (dihydroneopterin triphosphate) has been found in extracts of $\text{Drosophila melanogaster}$. NADP⁺ or NADPH and Mg²⁺ are required for this enzymatic transformation. No sepiapterin is produced when dihydroneopterin is supplied as substrate in place of dihydroneopterin triphosphate.     

Human deoxyuridine triphosphate nucleotidohydrolase. Purification and characterization of the deoxyuridine triphosphate nucleotidohydrolase from acute lymphocytic leukemia.

A new fast assay procedure for increasing deoxyuridine triphosphate nucleotidohydrolase activity was developed. With this assay procedure, this enzyme derived from blast cells of patients with acute lymphocytic leukemia was purified at least 1218-fold. The molecular weight was estimated by gel filtration to be 43,000. The enzyme exhibited optimal activity over a pH range of 7 to 8 and the activation energy was estimated to be 6.5 kcal/mol at pH 7.5. While the enzyme had activity in the absence of added divalent cations, the activity could be inhibited by EDTA but not by phenanthroline. The inhibition caused by EDTA could be reversed by Mg2+ or Zn2+. The enzyme had maximal activity in the presence of Mg2+ (40 muM) and Mg2+ (4 mM) stabilized the enzyme at 37 degrees C. Cupric ion (0.5 mM) inhibited (50%) enzyme activity in the presence or absence of Mg2+. The substrate for the enzyme was dUTP and the apparent Km was 1 muM. No other deoxyribonucleoside or ribonucleoside triphosphate served as a substrate for the enzyme.     

Ring opening is not rate-limiting in the GTP cyclohydrolase I reaction

GTP cyclohydrolase I catalyzes a mechanistically complex ring expansion affording dihydroneopterin triphosphate and formate from GTP. Single turnover quenched flow experiments were performed with the recombinant enzyme from Escherichia coli. The consumption of GTP and the formation of 5-formylamino-6-ribosylamino-2-amino-4(3H)-pyrimidinone triphosphate, formate, and dihydroneopterin triphosphate were determined by high pressure liquid chromatography analysis. A kinetic model comprising three consecutive unimolecular steps was used for interpretations where the first intermediate, 5-formylamino-6-ribosylamino-2-amino-4(3H)-pyrimidinone 5'-triphosphate, was formed in a reversible reaction. The rate constant k(1) for the reversible opening of the imidazole ring of GTP was 0.9 s(-1), the rate constant k(3) for the release of formate from 5-formylamino-6-ribosylamino-2-amino-4(3H)-pyrimidinone triphosphate was 2.0 s(-1), and the rate constant k(4) for the formation of dihydroneopterin triphosphate was 0.03 s(-1). Thus, the hydrolytic opening of the imidazole ring of GTP is rapid by comparison with the overall reaction.     

Reaction mechanism of GTP cyclohydrolase I: single turnover experiments using a kinetically competent reaction intermediate

GTP cyclohydrolase I catalyses the transformation of GTP into dihydroneopterin 3'-triphosphate, which is the first committed precursor of tetrahydrofolate and tetrahydrobiopterin. The kinetically competent reaction intermediate, 2-amino-5-formylamino-6-ribosylamino-4(3H)-pyrimidinone, was used as substrate for single turnover experiments monitored by multiwavelength photometry. The early reaction phase is characterized by the rapid appearance of an optical transient with an absorption maximum centred at 320. This species is likely to represent a Schiff base intermediate at the initial stage of the Amadori rearrangement of the carbohydrate side-chain. Deconvolution of the optical spectra suggested four linearly independent processes. A fifth reaction step was attributed to photodecomposition of the enzyme product. Pre-steady state experiments were also performed with the H179A mutant which can catalyse a reversible conversion of GTP to 2-amino-5-formylamino-6-ribosylamino-4(3H)-pyrimidinone but is unable to form the final product, dihydroneopterin triphosphate. Optical spectroscopy failed to detect any intermediate in the reversible reaction sequence catalysed by the mutant protein. The data obtained with the wild-type and mutant protein in conjunction with earlier quenched flow studies show that the enzyme-catalysed opening of the imidazole ring of GTP and the hydrolytic release of formate from the resulting formamide type intermediate are both rapid reactions by comparison with the subsequent rearrangement of the carbohydrate side-chain which precedes the formation of the dihydropyrazine ring of dihydroneopterin triphosphate.     

Crystallographic and kinetic investigations on the mechanism of 6-pyruvoyl tetrahydropterin synthase

The enzyme 6-pyruvoyl tetrahydropterin synthase (PTPS) catalyses the second step in the de novo biosynthesis of tetrahydrobiopterin, the conversion of dihydroneopterin triphosphate to 6-pyruvoyl tetrahydropterin. The Zn and Mg-dependent reaction includes a triphosphate elimination, a stereospecific reduction of the N5-C6 double bond and the oxidation of both side-chain hydroxyl groups. The crystal structure of the inactive mutant Cys42Ala of PTPS in complex with its natural substrate dihydroneopterinetriphosphate was determined at 1.9 A resolution. Additionally, the uncomplexed enzyme was refined to 2.0 A resolution. The active site of PTPS consists of the pterin-anchoring Glu A107 neighboured by two catalytic motifs: a Zn(II) binding site and an intersubunit catalytic triad formed by Cys A42, Asp B88 and His B89. In the free enzyme the Zn(II) is in tetravalent co-ordination with three histidine ligands and a water molecule. In the complex the water is replaced by the two substrate side-chain hydroxyl groups yielding a penta-co-ordinated Zn(II) ion. The Zn(II) ion plays a crucial role in catalysis. It activates the protons of the substrate, stabilizes the intermediates and disfavours the breaking of the C1'C2' bond in the pyruvoyl side-chain. Cys A42 is activated by His B89 and Asp B88 for proton abstraction from the two different substrate side-chain atoms C1', and C2'. Replacing Ala A42 in the mutant structure by the wild-type Cys by modelling shows that the C1' and C2' substrate side-chain protons are at equal distances to Cys A42 Sgamma. The basicity of Cys A42 may be increased by a catalytic triad His B89 and Asp B88. The active site of PTPS seems to be optimised to carry out proton abstractions from two different side-chain C1' and C2' atoms, with no obvious preference for one of them. Kinetic studies with dihydroneopterin monophosphate reveal that the triphosphate moiety of the substrate is necessary for enzyme specifity.     

Synthesis, utilization, and structure of the tetrahydropterin intermediates in the bovine adrenal medullary de novo biosynthesis of tetrahydrobiopterin

The biosynthesis of two tetrahydropterin intermediates (H4pterin-1 and H4pterin-2), their conversion to tetrahydrobiopterin, and their overall chemical structures are described. A new high performance liquid chromatographic separation of these and other tetrahydropterins is also described. The biosynthesis of tetrahydrobiopterin from dihydroneopterin triphosphate proceeds in the presence of the bovine adrenal medullary biosynthetic enzymes, Mg2+, NADPH. The biosynthesis of H4pterin-2 occurs under identical conditions, and the compound accumulates in the presence of 1 to 10 microM of N-acetylserotonin, an inhibitor of sepiapterin reductase. At higher concentrations of the inhibitor, the synthesis of H4pterin-2 is also inhibited, and H4pterin-1 accumulates. H4pterin-1 also accumulates in the absence of NADPH. In the presence of NADPH the biosynthetic enzymes convert both intermediates to tetrahydrobiopterin at rates which are greater than the rate of conversion of dihydroneopterin triphosphate to tetrahydrobiopterin. Electrochemical, UV/VIS, oxidation, and ionization properties identify the compounds as tetrahydropterins. The side chain structures of the compounds were determined by a combination of chemical means. The structures of the compounds are 6R-(1',2'-dioxopropyl)-tetrahydropterin (H4pterin-1) and 6R-(L-1'-hydroxy-2'-oxopropyl)-tetrahydropterin (H4pterin-2). The data indicate that the biosynthesis of tetrahydrobiopterin from dihydroneopterin triphosphate proceeds in three steps: 1) formation of H4pterin-1 in the presence of Mg2+, 2) NADPH-dependent conversion of H4pterin-1 to H4pterin-2, and 3) NADPH-dependent conversion of H4pterin-2 to tetrahydrobiopterin.