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PubMed2001

7,8-Dihydropterin-6-carboxylic acid as light emitter of luminous millipede, Luminodesmus sequoiae.

A luminous millipede. Luminodesmus sequoiae, emits light centered at a wavelength of 500 nm. To determine the light emitter of this bioluminescent system, fluorescent compounds were isolated from pulverized cuticles. NMR and MS spectra of these compounds showed them to be pterin derivatives. Furthermore, proton/deuterium (H/D) exchange experiments by ESI-Q-TOF-MS and -MS/MS measurements have proved to be a powerful tool for elucidating these heteroaromatic compounds. Finally, we have concluded that 7,8-dihydropterin-6-carboxylic acid, a new natural product, is the light emitter of Luminodesmus bioluminescence.

Animals↗
PubMed2000

A sensitive method to assay the xanthine oxidase activity in primary cultures of cerebellar granule cells.

Since xanthine oxidase (XO, Xanthine:oxidoreductase, E.C.1.2.3.22) is a key enzyme in reactive oxygen specie formation which plays a major role in cell oxidative stress, the availability of a sensitive and simple assay useful to detect its activity in monolayer cell cultures is worthwhile. In order to achieve this, we developed a method in which the conversion of pterine into isoxanthopterin is monitored fluorimetrically. Temperature assay was 50 degrees C. The activity of XO was detected in cerebellar granule cells exposed to glutamate. Since XO is formed from protease-dependent xanthine dehydrogenase processing, its activity appearance was found to be prevented by the protease inhibitor, leupeptin, as well as the glutamate NMDA-receptor inhibitor, MK-801, and the Ca(++) complexing agent, EGTA. The reported novel protocol, at variance with a conventional method, is shown to be a simple, fast, sensitive and relatively cheap method to assay XO activity. In addition, the reported assay can be applied to any cell type in culture.

Animals↗
PubMed2000

Low-temperature optical absorption spectra suggest a redox role for tetrahydrobiopterin in both steps of nitric oxide synthase catalysis.

To investigate the role of tetrahydrobiopterin (BH4) in the catalytic mechanism of nitric oxide synthase (NOS), we analyzed the spectral changes following addition of oxygen to the reduced oxygenase domain of endothelial nitric oxide synthase (NOS) in the presence of different pteridines at -30 degrees C. In the presence of N(G)-hydroxy-L-arginine (NOHLA) and BH4 or 5-methyl-BH4, both of which support NO synthesis, the first observable species were mixtures of high-spin ferric NOS (395 nm), ferric NO-heme (439 nm), and the oxyferrous complex (417 nm). With Arg, no clear intermediates could be observed under the same conditions. In the presence of the BH4-competitive inhibitor 7,8-dihydrobiopterin (BH2), intermediates with maxima at 417 and 425 nm were formed in the presence of Arg and NOHLA, respectively. In the presence of 4-amino-BH4, the maxima of the intermediates with Arg and NOHLA were at 431 and 423 nm, respectively. We ascribe all four spectra to oxyferrous heme complexes. The intermediates observed in this study slowly decayed to the high-spin ferric state at -30 degrees C, except for those formed in the presence of 4-amino-BH4, which required warming to room temperature for regeneration of high-spin ferric NOS; with Arg, regeneration remained incomplete. From these observations, we draw several conclusions. (1) BH4 is required for reductive oxygen activation, probably as a transient one-electron donor, not only in the reaction with Arg but also with NOHLA; (2) in the absence of redox-active pterins, reductive oxygen activation does not occur, which results in accumulation of the oxyferrous complex; (3) the spectral properties of the oxyferrous complex are affected by the presence and identity of the substrate; (4) the slow and incomplete formation of high-spin ferric heme with 4-amino-BH4 suggests a structural cause for inhibition of NOS activity by this pteridine.

Animals↗
PubMed1983

6,6-Dimethylpterins: stable quinoid dihydropterin substrate for dihydropteridine reductase and tetrahydropterin cofactor for phenylalanine hydroxylase.

The tautomeric structure of the cofactor product of aromatic amino acid hydroxylases, quinoid dihydrobiopterin, is still unknown. Characterization of this molecule, which is also the substrate for dihydropteridine reductase (EC 1.6.99.7), has been hindered by the rapid rearrangement of quinoid dihydropterins to 7,8-dihydropterins. This tautomerization can be prevented by disubstitution at the 6-position. A procedure is presented for the synthesis of 6,6-disubstituted pterins from a vicinal diamine and 2-amino-6-chloro-4(3H)-pyrimidinone. The method is illustrated with the specific synthesis of 6,6-dimethyltetrahydropterin (6,6-Me2PH4). 6,6-Me2PH4 is a cofactor for rat liver phenylalanine hydroxylase (EC 1.14.16.1), with enzyme kinetic parameters similar to those of its positional isomer, 6,7-dimethyltetrahydropterin. The resulting quinoid 6,6-dimethyldihydropterin (q-6,6-Me2PH2) is stable; the half-life in 0.1 M Tris-HCl, pH 7.4, at 27 and 37 degrees C is 4 and 1.25 h, respectively. q-6,6-Me2PH2, produced either by phenylalanine hydroxylase or by chemical oxidation of 6,6-Me2PH4, is a substrate for dihydropteridine reductase, with a Km of 0.4 mM and a maximum velocity double that of the natural isomer of quinoid dihydrobiopterin. In concentrations up to 0.4 mM q-6,6-Me2PH2 is not an inhibitor of phenylalanine hydroxylase, in contrast to 6-methyl-7,8-dihydropterin and 7,8-dihydrobiopterin which inhibit competitively, with Ki's of 0.2 mM and 0.05 mM, respectively. The stability of q-6,6-Me2PH2 has facilitated definitive determination of chemical and physical properties of a quinoid dihydropterin.

Animals↗
PubMed1999

Purification and characterization of a tungsten-containing formate dehydrogenase from Desulfovibrio gigas.

An air-stable formate dehydrogenase (FDH), an enzyme that catalyzes the oxidation of formate to carbon dioxide, was purified from the sulfate reducing organism Desulfovibrio gigas (D. gigas) NCIB 9332. D. gigas FDH is a heterodimeric protein [alpha (92 kDa) and beta (29 kDa) subunits] and contains 7 +/- 1 Fe/protein and 0.9 +/- 0.1 W/protein. Selenium was not detected. The UV/visible absorption spectrum of D. gigas FDH is typical of an iron-sulfur protein. Analysis of pterin nucleotides yielded a content of 1.3 +/- 0.1 guanine monophosphate/mol of enzyme, which suggests a tungsten coordination with two molybdopterin guanine dinucleotide cofactors. Both Mössbauer spectroscopy performed on D. gigas FDH grown in a medium enriched with (57)Fe and EPR studies performed in the native and fully reduced state of the protein confirmed the presence of two [4Fe-4S] clusters. Variable-temperature EPR studies showed the presence of two signals compatible with an atom in a d(1) configuration albeit with an unusual relaxation behavior as compared to the one generally observed for W(V) ions.

Aerobiosis↗
PubMed2003

Uncoupled forms of tyrosine hydroxylase unmask kinetic isotope effects on chemical steps.

Tyrosine hydroxylase (TyrH) catalyzes the hydroxylation of tyrosine to dihydroxyphenylalanine. In the proposed mechanism, a ferryl-oxo species attacks the aromatic ring of tyrosine, forming a cationic intermediate. However, no significant isotope effect is found for wild-type TyrH when 3,5-2H2-tyrosine is used as a substrate. The isotope effect has now been determined with 3,5-2H2-tyrosine using mutant forms of TyrH in which the oxidation of the pterin is uncoupled from hydroxylation of the amino acid. Three mutant enzymes exhibit significant inverse deuterium isotope effects and inverse solvent isotope effects. A proton inventory for the E326A enzyme is consistent with a normal solvent isotope effect of 2.4 on an unproductive step. The results support the proposed mechanism and demonstrate the utility of using mutant proteins with branched pathways to reveal isotope effects which are masked in the wild-type enzyme.

Deuterium Exchange Measurement↗
PubMed2001

Bisubstrate analogue inhibitors of 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase: synthesis and biochemical and crystallographic studies.

6-Hydroxymethyl-7,8-dihydropterin pyrophosphokinase (HPPK) catalyzes the transfer of pyrophosphate from ATP to 6-hydroxymethyl-7,8-dihydropterin (HP), leading to the biosynthesis of folate cofactors. Like other enzymes in the folate pathway, HPPK is an ideal target for the development of antimicrobial agents because the enzyme is essential for microorganisms but is absent from human and animals. Three bisubstrate analogues have been synthesized for HPPK and characterized by biochemical and X-ray crystallographic analyses. All three bisubstrate analogues consist of a pterin, an adenosine moiety, and a link composed of 2-4 phosphoryl groups. P(1)-(6-Hydroxymethylpterin)-P(2)-(5'-adenosyl)diphosphate (HP(2)A, 5) shows little affinity and inhibitory activity for E. coli HPPK. P(1)-(6-Hydroxymethylpterin)-P(3)-(5'-adenosyl)triphosphate (HP(3)A, 6) shows moderate affinity and inhibitory activity with K(d) = 4.25 microM in the presence of Mg(2+) and IC(50) = 1.27 microM. P(1)-(6-Hydroxymethylpterin)-P(4)-(5'-adenosyl)tetraphosphate (HP(4)A, 7) shows the highest affinity and inhibitory activity with K(d) = 0.47 microM in the presence of Mg(2+) and IC(50) = 0.44 microM. The affinity of MgHP(4)A for HPPK is approximately 116 and 76 times higher than that of MgADP and 6-hydroxymethylpterin, respectively. The crystal structure of HPPK in complex with 7 (HPPK.MgHP(4)A) has been determined at 1.85 A resolution with a crystallographic R factor of 0.185. The crystal structure shows that 7 occupies both HP- and ATP-binding sites and induces significant conformational changes in HPPK. The biochemical and structural studies of the bisubstrate analogues indicate that the bisubstrate analogue approach can produce more potent inhibitors for HPPK and the minimum length of the link for a bisubstrate analogue is approximately 7 A.

Adenosine Triphosphate↗
PubMed1989

Effects of sepiapterin and 6-acetyldihydrohomopterin on the guanosine triphosphate cyclohydrolase I of mouse, rat and the fruit-fly Drosophila.

The regulation of GTP cyclohydrolase I would lead to the regulation of tetrahydrobiopterin, an important cofactor for synthesis of neurotransmitters. In an attempt to extend a previous finding [Bellahsene, Dhondt, & Farriaux (1984) Biochem. J. 217, 59-65] that GTP cyclohydrolase I of rat liver is inhibited by subnanomolar concentrations of reduced biopterin and sepiapterin, we found that this could not be verified with the enzyme from mouse liver, fruit-fly (Drosophila) heads or, indeed, from rat liver. It was shown, however, that 12 microM-sepiapterin inhibited mouse liver GTP cyclohydrolase I. Another compound, namely 6-acetyldihydrohomopterin, was also employed in the present study to explore its effect on enzymes that lead to its synthesis in Drosophila and for effects on mammalian systems; at 2-5 microM this compound was shown to stimulate one form of mouse liver GTP cyclohydrolase I and then to inhibit at higher concentrations (40 microM). Neither sepiapterin nor 6-acetyldihydrohomopterin caused any effect on the Drosophila head enzyme. On the other hand, the sigmoid GTP concentration curve for the Drosophila enzyme may indicate a regulatory characteristic of this enzyme. Another report, on the lower level of GTP cyclohydrolase I in mutant mouse liver [McDonald, Cotton, Jennings, Ledley, Woo & Bode (1988) J. Neurochem. 50, 655-657], was confirmed and extended. Instead of having 10% activity, we find that the hph-1 mouse mutant has less than 2% activity in the liver. These studies demonstrate that micromolar levels of reduced pterins may have regulatory effects on GTP cyclohydrolase I and that a mouse mutant is available that has low enough activity to be considered as a model for human atypical phenylketonuria.

Aminohydrolases↗
PubMed1996

Neurochemical effects following peripheral administration of tetrahydropterin derivatives to the hph-1 mouse.

The hph-1 mouse which displays tetrahydrobiopterin deficiency and impaired dopamine and serotonin turnover, has been used to study cofactor replacement therapy for disorders causing brain tetrahydrobiopterin deficiency. Subcutaneous administration of 100 mumol/kg (30 mg/kg) of tetrahydrobiopterin resulted in a twofold increase in brain cofactor concentration 1 h after administration. Concentrations remained above the endogenous level for at least 4 h but returned to normal by 24 h. The lipophilic tetrahydrobiopterin analogue 6-methyltetrahydropterin entered the brain five times more efficiently than tetrahydrobiopterin but was cleared at a faster rate. Tetrahydropterins linked to the lipoidal carrier N-benzyl-1, 4-dihydronicotinoyl did not result in a detectable increase in levels of brain pterins over the period of the study (1-4 h). Stimulation of monoamine turnover was not observed at any time point with either natural cofactor or the methyl analogue. Increasing the amount of tetrahydrobiopterin to 1,000 mumol/kg resulted in elevation of cofactor concentrations, a brief increase in the activity of tyrosine and tryptophan hydroxylase 1 h postadministration, and increased turnover of dopamine and serotonin metabolites lasting 24 h. However, 2 of 12 (17%) mice died following administration of this dose of cofactor. Our findings suggest that acute peripheral tetrahydrobiopterin administration is unlikely to stimulate brain monoamine turnover directly unless very large and potentially toxic doses of cofactor are used.

Animals↗
PubMed1983

Biosynthesis of tetrahydrobiopterin by de novo and salvage pathways in adrenal medulla extracts, mammalian cell cultures, and rat brain in vivo.

Mammalian cells and tissues were found to have two pathways for the biosynthesis of tetrahydrobiopterin (BH4): (i) the conversion of GTP to BH4 by a methotrexate-insensitive de novo pathway, and (ii) the conversion of sepiapterin to BH4 by a pterin salvage pathway dependent on dihydrofolate reductase (5,6,7,8-tetrahydrofolate: NADP+ oxidoreductase, EC 1.5.1.3) activity. In a Chinese hamster ovary cell mutant lacking dihydrofolate reductase (DUKX-B11), endogenous formation of BH4 proceeds normally but, unlike the parent cells, these cells or extracts of them do not convert sepiapterin or 7,8-dihydrobiopterin to BH4. KB cells, which do not contain detectable levels of GTP cyclohydrolase or BH4 but do contain dihydrofolate reductase, readily convert sepiapterin to BH4 and this conversion is completely prevented by methotrexate. In supernatant fractions of bovine adrenal medulla, the conversion of sepiapterin to BH4 is completely inhibited by methotrexate. Similarly, this conversion in rat brain in vivo is methotrexate-sensitive. Sepiapterin and 7,8-dihydrobiopterin apparently do not enter the de novo pathway of BH4 biosynthesis and may be derived from labile intermediates which have not yet been characterized.

Adrenal Medulla↗
PubMed1991

Localization of cofactor binding sites with monoclonal anti-idiotype antibodies: phenylalanine hydroxylase.

A monoclonal anti-idiotype antibody, NS7, previously shown to mimic the binding of the pterin cofactor of phenylalanine hydroxylase (phenylalanine 4-monooxygenase, EC 1.14.16.1) has been used to localize the cofactor binding site within the phenylalanine hydroxylase catalytic domain to a 27-amino-acid sequence that is highly conserved among the three aromatic amino acid hydroxylases. The binding of NS7 to a synthetic peptide corresponding to the phenylalanine hydroxylase sequence from residue 263 to residue 289 was blocked by the competitive inhibitor of phenylalanine hydroxylase enzyme activity, 7,8-dihydro-6,7-dimethylpterin. In addition this peptide competed with native phenylalanine hydroxylase for binding to 6,7-dimethyl-5,6,7,8-tetrahydropterin conjugated to a polyglutamate carrier. Application of this simple and direct approach to other enzymes is likely to greatly facilitate the identification of ligand binding sites on enzymes, which will significantly contribute to the understanding of enzyme structure-function relationships.

Amino Acid Sequence↗
PubMed1999

A corrinoid-dependent catabolic pathway for growth of a Methylobacterium strain with chloromethane.

Methylobacterium sp. strain CM4, an aerobic methylotrophic alpha-proteobacterium, is able to grow with chloromethane as a carbon and energy source. Mutants of this strain that still grew with methanol, methylamine, or formate, but were unable to grow with chloromethane, were previously obtained by miniTn5 mutagenesis. The transposon insertion sites in six of these mutants mapped to two distinct DNA fragments. The sequences of these fragments, which extended over more than 17 kb, were determined. Sequence analysis, mutant properties, and measurements of enzyme activity in cell-free extracts allowed the definition of a multistep pathway for the conversion of chloromethane to formate. The methyl group of chloromethane is first transferred by the protein CmuA (cmu: chloromethane utilization) to a corrinoid protein, from where it is transferred to H4folate by CmuB. Both CmuA and CmuB display sequence similarity to methyltransferases of methanogenic archaea. In its C-terminal part, CmuA is also very similar to corrinoid-binding proteins, indicating that it is a bifunctional protein consisting of two domains that are expressed as separate polypeptides in methyl transfer systems of methanogens. The methyl group derived from chloromethane is then processed by means of pterine-linked intermediates to formate by a pathway that appears to be distinct from those already described in Methylobacterium. Remarkable features of this pathway for the catabolism of chloromethane thus include the involvement of a corrinoid-dependent methyltransferase system for dehalogenation in an aerobe and a set of enzymes specifically involved in funneling the C1 moiety derived from chloromethane into central metabolism.

Amino Acid Sequence↗
PubMed2004

Regioselective nitration of tryptophan by a complex between bacterial nitric-oxide synthase and tryptophanyl-tRNA synthetase.

Bacterial nitric-oxide synthase proteins (NOSs) from certain Streptomyces strains have been shown to participate in biosynthetic nitration of tryptophanyl moieties in vivo (Kers, J. A., Wach, M. J., Krasnoff, S. B., Cameron, K. D., Widom, J., Bukhaid, R. A., Gibson, D. M., and Crane, B. R., and Loria, R. (2004) Nature 429, 79-82). We report that the complex between Deinococcus radiodurans NOS (deiNOS) and an unusual tryptophanyl-tRNA synthetase (TrpRS II) catalyzes the regioselective nitration of tryptophan (Trp) at the 4-position. Unlike non-enzymatic Trp nitration, and similar reactions catalyzed by globins and peroxidases, deiNOS only produces the otherwise unfavorable 4-nitro-Trp isomer. Although deiNOS alone will catalyze 4-nitro-Trp production, yields are significantly enhanced by TrpRS II and ATP. 4-Nitro-Trp formation exhibits saturation behavior with Trp (but not tyrosine) and is completely inhibited by the addition of the mammalian NOS cofactor (6R)-5,6,7,8-tetrahydro-l-biopterin (H(4)B). Trp stimulates deiNOS oxidation of substrate l-arginine (Arg) to the same degree as H(4)B. These observations are consistent with a mechanism where Trp or a derivative thereof binds in the NOS pterin site, participates in Arg oxidation, and becomes nitrated at the 4-position.

Deinococcus↗
PubMed2001

L-ascorbic acid potentiates endothelial nitric oxide synthesis via a chemical stabilization of tetrahydrobiopterin.

Ascorbic acid has been shown to stimulate endothelial nitric oxide (NO) synthesis in a time- and concentration-dependent fashion without affecting NO synthase (NOS) expression or l-arginine uptake. The present study investigates if the underlying mechanism is related to the NOS cofactor tetrahydrobiopterin. Pretreatment of human umbilical vein endothelial cells with ascorbate (1 microm to 1 mm, 24 h) led to an up to 3-fold increase of intracellular tetrahydrobiopterin levels that was concentration-dependent and saturable at 100 microm. Accordingly, the effect of ascorbic acid on Ca(2+)-dependent formation of citrulline (co-product of NO) and cGMP (product of the NO-activated soluble guanylate cyclase) was abolished when intracellular tetrahydrobiopterin levels were increased by coincubation of endothelial cells with sepiapterin (0.001-100 microm, 24 h). In contrast, ascorbic acid did not modify the pterin affinity of endothelial NOS, which was measured in assays with purified tetrahydrobiopterin-free enzyme. The ascorbate-induced increase of endothelial tetrahydrobiopterin was not due to an enhanced synthesis of the compound. Neither the mRNA expression of the rate-limiting enzyme in tetrahydrobiopterin biosynthesis, GTP cyclohydrolase I, nor the activities of either GTP cyclohydrolase I or 6-pyruvoyl-tetrahydropterin synthase, the second enzyme in the de novo synthesis pathway, were altered by ascorbate. Our data demonstrate that ascorbic acid leads to a chemical stabilization of tetrahydrobiopterin. This was evident as an increase in the half-life of tetrahydrobiopterin in aqueous solution. Furthermore, the increase of tetrahydrobiopterin levels in intact endothelial cells coincubated with cytokines and ascorbate was associated with a decrease of more oxidized biopterin derivatives (7,8-dihydrobiopterin and biopterin) in cells and cell supernatants. The present study suggests that saturated ascorbic acid levels in endothelial cells are necessary to protect tetrahydrobiopterin from oxidation and to provide optimal conditions for cellular NO synthesis.

Ascorbic Acid↗
PubMed2003

The autoxidation of tetrahydrobiopterin revisited. Proof of superoxide formation from reaction of tetrahydrobiopterin with molecular oxygen.

It has been known for quite some time that tetrahydrobiopterin (H4B) is prone to autoxidation in the presence of molecular oxygen. Evidence has been presented that in this process superoxide radicals may be released, although their intermediacy never has been directly proven. In the present study, the autoxidation of H4B was reinvestigated with the aim to find direct evidence for superoxide formation. By means of two specific assays, namely elicitation of luminescence from lucigenin and ESR-spectrometric detection of the DEPMPO-OOH radical adduct, the release of free superoxide radicals was unequivocally demonstrated. The production of superoxide radicals was further corroborated by interaction with nitric oxide. The kinetics of the autoxidation process was established. Our data fully confirm earlier conclusions that the direct reaction between H4B and oxygen serves as an initiation reaction for the further, rapid reaction of the thus formed superoxide with H4B, thereby very likely establishing a chain reaction process involving reduction of molecular oxygen by the intermediary tetrahydrobiopterin radical. Conclusively, because H4B can per se induce oxidative stress, an in vivo overproduction of this pterin, as is evident in various diseases, may be responsible for the observed acceleration of pathophysiological pathways.

Biopterins↗
PubMed2003

Catalytic and thermodynamic properties of tetrahydromethanopterin-dependent serine hydroxymethyltransferase from Methanococcus jannaschii.

The reaction catalyzed by serine hydroxymethyltransferase (SHMT), the transfer of Cbeta of serine to tetrahydropteroylglutamate, represents in Eucarya and Eubacteria a major source of one-carbon (C1) units for several essential biosynthetic processes. In many Archaea, C1 units are carried by modified pterin-containing compounds, which, although structurally related to tetrahydropteroylglutamate, play a distinct functional role. Tetrahydromethanopterin, and a few variants of this compound, are the modified folates of methanogenic and sulfate-reducing Archaea. Little information on SHMT from Archaea is available, and the metabolic role of the enzyme in these organisms is not clear. This contribution reports on the purification and characterization of recombinant SHMT from the hyperthermophilic methanogen Methanococcus jannaschii. The enzyme was characterized with respect to its catalytic, spectroscopic, and thermodynamic properties. Tetrahydromethanopterin was found to be the preferential pteridine substrate. Tetrahydropteroylglutamate could also take part in the hydroxymethyltransferase reaction, although with a much lower efficiency. The catalytic features of the enzyme with substrate analogues and in the absence of a pteridine substrate were also very similar to those of SHMT isolated from Eucarya or Eubacteria. On the other hand, the M. jannaschii enzyme showed increased thermoactivity and resistance to denaturating agents with respect to the enzyme purified from mesophilic sources. The results reported suggest that the active site structure and the mechanism of SHMT are conserved in the enzyme from M. jannaschii, which appear to differ only in its ability to bind and use a modified folate as substrate and increased thermal stability.

Amino Acid Sequence↗
PubMed2006

Chemical natures and application of 6-formylpterin derivatives.

Pterin, an analog of guanine, is an electron transfer compound in biological systems. Among the analogs, 6-formylpterin (6FP) has been demonstrated to have many marked physiological and pharmacological activities. In vitro, 6FP converts molecular oxygen to reactive oxygen species (ROS) in the presence of NADH or NADPH under light illumination, with the oxidation of NADH is to NAD(+). In the present study, it has been elucidated that some of 6FP derivatives in which the 3-position of 6FP is modified have such unique activities even in the dark where the most of in vivo biological events occur.

Darkness↗
PubMed2001

Isolation and purification of tyrosine hydroxylase from callus cultures of Portulaca grandiflora.

Tyrosine hydroxylase was separated from polyphenol oxidase activity and was highly purified from betacyanin producing callus cultures of Portulaca grandiflora. The purified enzyme catalyzed the formation of DOPA (L-3,4-dihydroxyphenylalanine) from tyrosine and required the pterin compounds (6-methyl-5,6,7,8-tetrahydropterin; 5,6,7,8-tetrahydrobiopterin; 6,7-dimethyl-5,6,7,8-tetrahydropterin) as coenzyme. The K(m) values for tyrosine and 6-methyl-5,6,7,8-tetrahydropterin were 0.5 mM and 0.15 mM, respectively. This enzyme was activated by Fe(2+) and Mn(2+), and inhibited by metal chelating agents.

Catalysis↗