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PubMed2000

The aromatic amino acid hydroxylases.

The enzymes phenylalanine hydroxylase, tyrosine hydroxylase, and tryptophan hydroxylase constitute the family of pterin-dependent aromatic amino acid hydroxylases. Each enzyme catalyzes the hydroxylation of the aromatic side chain of its respective amino acid substrate using molecular oxygen and a tetrahydropterin as substrates. Recent advances have provided insights into the structures, mechanisms, and regulation of these enzymes. The eukaryotic enzymes are homotetramers comprised of homologous catalytic domains and discrete regulatory domains. The ligands to the active site iron atom as well as residues involved in substrate binding have been identified from a combination of structural studies and site-directed mutagenesis. Mechanistic studies with nonphysiological and isotopically substituted substrates have provided details of the mechanism of hydroxylation. While the complex regulatory properties of phenylalanine and tyrosine hydroxylase are still not fully understood, effects of regulation on key kinetic parameters have been identified. Phenylalanine hydroxylase is regulated by an interaction between phosphorylation and allosteric regulation by substrates. Tyrosine hydroxylase is regulated by phosphorylation and feedback inhibition by catecholamines.

Amino Acids, Cyclic↗

PubMed2005

Total truncation of the molybdopterin/dimerization domains of SUOX protein in an Arab family with isolated sulfite oxidase deficiency.

We ascertained a patient with the full-blown phenotype of isolated sulfite oxidase deficiency in a consanguineous Arab family. The proband's phenotype included the presence of intractable seizures in the neonatal period, some dysmorphic features, neuroradiologic findings reminiscent of hypoxic ischemic encephalopathy and rapidly progressive brain destruction leading to severe neurodevelopmental impairment. Biochemically, the patient excreted a large amount of S-sulfocysteine with normal amounts of xanthene and hypoxanthine and had normal plasma uric acid, which was consistent with isolated sulfite oxidase deficiency. We report the identification of the first Arab mutation in SUOX, the gene for sulfite oxidase enzyme, in the ascertained family. The newly identified Arab mutation in the SUOX gene (a single nucleotide deletion, del G1244) is predicted to cause a frame shift at amino acid 117 of the translated protein with the generation of a stop codon and total truncation of the molybdo-pterin- and the dimerizing-domain(s) of SUOX protein expressed from the mutant allele. The identification of this new Arab SUOX mutation should facilitate pre-implantation genetic diagnosis and selection of unaffected embryos for future pregnancy in the ascertained family with the mutation and related families with the same mutation.

Abnormalities, Multiple↗

PubMed2003

Mechanism of aromatic hydroxylation by an activated FeIV=O core in tetrahydrobiopterin-dependent hydroxylases.

The chemical pathways leading to the hydroxylated aromatic amino acids in phenylalanine and tryptophan hydroxylases have been investigated by means of hybrid density functional theory. In the catalytic core of these non-heme iron enzymes, dioxygen reacts with the pterin cofactor and is likely to be activated by forming an iron(IV)=O complex. The capability of this species to act as a hydroxylating intermediate has been explored. Depending on the protonation state of the ligands of the metal, two different mechanisms are found to be energetically possible for the hydroxylation of phenylalanine and tryptophan by the high-valent iron-oxo species. With a hydroxo ligand the two-electron oxidation of the aromatic ring passes through a radical, while an arenium cation is involved when a water replaces the hydroxide. After the attack of the activated oxygen on the substrate, it is also found that a 1,2-hydride shift (known as an NIH shift) generates a keto intermediate, which can decay to the true product through an intermolecular keto-enol tautomerization. The benzylic hydroxylation of 4-methylphenylalanine by the Fe(IV)=O species has also been investigated according to the rebound mechanism. The computed energetics lead to the conclusion that Fe(IV)=O is capable not only of aromatic hydroxylation, but also of benzylic hydroxylation.

Amino Acids, Aromatic↗

PubMed2004

Self-assembly of folic acid derivatives: induction of supramolecular chirality by hierarchical chiral structures.

Hierarchical chiral structures made up of dendritic oligo(L- or D-glutamic acid) moieties of folic acid derivatives induce supramolecular chirality in the self-assembled columnar structures of the folic acids. These folic acids self-assemble through the intermolecular hydrogen bonds of the pterin rings to form disklike tetramers. In the neat states, the stacked tetramers form thermotropic hexagonal columnar phases over wide temperature ranges, including room temperature. Addition of alkali metal salts induces chirality in the columnar phases. In dilute solution states in a relatively polar solvent (chloroform), the folic acid derivatives form non-chiral, self-assembled structures. In the presence of sodium triflate, the folic acid forms chiral columnar assemblies through the oligo(L-glutamic acid) moiety, similar to those formed in the liquid-crystalline (LC) states. The enantiomer of the folic acid induces columnar assemblies with reversed helicity. In the case of the diastereomer, no induced helicity is observed. Application of an apolar solvent (dodecane) drives the folic acid derivatives to form chiral assemblies in the absence of ions. In this case, lipophilic interactions promote nanophase segregation, which enhances the formation of chiral columns. Interestingly, the chiral supramolecular structure of the diastereomer induces the most intense circular dichroism. In both cases, the molecular chirality in the oligo(glutamate) moieties yields supramolecular chirality of the folic acids that self-assemble through cooperative molecular interactions.

Journal Article↗

PubMedDate not supplied

Analysis of photoreceptor proteins of microorganisms by gradient gel electrophoresis and other biochemical separation methods.

Photoreceptor proteins for photoorientation in microorganisms are usually membrane bound and can be isolated by standard biochemical methods. Three examples are shown: the flagellates Euglena gracilis, Peridinum gatunense and the slime mold Dictyostelium discoideum. The photoreceptor of Euglena is attached to the basis of the flagellum and is composed of at least four chromoproteins which can be separated by gradient sodium dodecyl sulfate - polyacrylamide gel electrophoresis (SDS-PAGE) fast protein liquid chromatography (FPLC) and isoelectric focusing (IEF); it contains pterins and a flavin as chromophoric groups. The photoreceptor of Peridinium absorbs in the red wavelength band. Though not yet identified in detail, multiple receptors are probably involved, as indicated by fluorescence spectroscopy. Dictyostelium shows positive and negative phototaxis in its amoebal form and exclusively positive phototaxis in its pseudoplasmodial form. It is still open to discussion whether the two stages use separate photoreceptors. From amoebae two photoreceptor pigments have been isolated, showing an absorption which resembles the action spectrum, one membrane bound with a molecular mass of 45 kDa and one cytoplasmic fraction with a molecular mass of 27 kDa.

Animals↗

PubMed2003

Mutations in the molybdenum cofactor biosynthetic genes MOCS1, MOCS2, and GEPH.

Molybdenum cofactor deficiency in humans results in the loss of the activity of molybdoenzymes sulfite oxidase, xanthine dehydrogenase, and aldehyde oxidase. The resultant severe phenotype, which includes progressive neurological damage leading in most cases to early childhood death, results primarily from the deficiency of sulfite oxidase. All forms of molybdenum cofactor deficiency are inherited as autosomal recessive traits. The cofactor is an unstable reduced pterin with a unique four-carbon side chain, synthesized by a complex pathway that requires the products of at least four different genes (MOCS1, MOCS2, MOCS3, and GEPH). Disease-causing mutations have been identified in three of these genes: MOCS1, MOCS2, and GEPH. MOCS1 and MOCS2 have a bicistronic architecture; i.e., each gene encodes two proteins in different open reading frames. The protein products, MOCS1A and B and MOCS2A and B, are expressed either from different mRNAs generated by alternative splicing or by independent translation of a bicistronic mRNA. The gephyrin protein, encoded by a third locus, is required during cofactor assembly for insertion of molybdenum. A total of 32 different disease-causing mutations, including several common to more than one family, have been identified in molybdenum cofactor-deficient patients and their relatives.

Animals↗

PubMed1995

Three-dimensional structure of the bifunctional protein PCD/DCoH, a cytoplasmic enzyme interacting with transcription factor HNF1.

The bifunctional protein pterin-4a-carbinolamine dehydratase (PCD)/dimerization cofactor of HNF1 (DCoH) is a cytoplasmic enzyme involved in the tetrahydrobiopterin regeneration and is found in complex with the transcription factor HNF1 in liver cell nuclei. An atypical hyperphenylalaninemia and the depigmentation disorder vitiligo are related to a deficiency of PCD/DCoH activity. The crystal structure of PCD/DCoH was solved by multiple isomorphous replacement and refined to a crystallographic R-factor of 20.5% at 2.7 A resolution. The single domain monomer comprises three alpha-helices packed against one side of a four-stranded, antiparallel beta-sheet. The functional enzyme is a homo-tetramer of 222 symmetry where each of the monomers contributes one helix to a central four helix bundle. In the tetramer two monomers form an eight-stranded, antiparallel beta-sheet with six helices packing against it from one side. The concave, hydrophobic surface of the eight-stranded beta-sheet with its two protruding loops at either end is reminiscent of the saddle-like shape seen in the TATA-box binding protein. PCD/DCoH binds as a dimer to the helical dimerization domain of dimeric HNF1 forming a hetero-tetramer possibly through a mixed four helix bundle.

Amino Acid Sequence↗

PubMed1990

Analysis of the tetrahydrobiopterin synthesizing system during maturation of murine reticulocytes.

The enzymes of tetrahydrobiopterin synthesis have been studied in murine bone marrow, in spleen, in erythrocytes, and in reticulocytes. Mice with chemically induced and with genetically conditioned reticulocytosis as found in the lactate dehydrogenase deficient strain (Ldh-1c/Ldh-1c) were used for analysis of reticulocytic enzyme activities. The activity of the biopterin synthesizing system is highest in bone marrow even though it amounts to only about 10% as compared with liver. The first enzyme of the biosynthetic pathway, GTP-cyclohydrolase, virtually disappears during the final maturation step of reticulocytes. In contrast, the activities of 6-pyruvoyltetrahydropterin synthase and of sepiapterin reductase of erythrocytes are only reduced to about one half of the reticulocyte level. The absence of biopterin in erythrocytes is therefore caused by the loss of the enzyme that initiates the pterin biosynthetic pathway.

Alcohol Oxidoreductases↗

PubMed2002

Further case of paroxysmal exercise-induced dystonia and some insights into pathogenesis.

Cerebrospinal fluid (CSF) analysis of pterin and monamine metabolites was performed before and after an attack in a patient with paroxysmal exercise-induced dystonia. A twofold increase in CSF homovanillic acid and 5-hydroxyindoleacetic acid after an attack was measured. This finding lends support to the hypothesis that increased dopaminergic transmission contributes to the clinical features of the hyperkinetic movement disorders.

Adult↗

PubMed2004

Biology and chemistry of the inhibition of nitric oxide synthases by pteridine-derivatives as therapeutic agents.

Inhibitors of the family of nitric oxide synthases (NOS-I-III; EC 1.14.13.39) are of interest as pharmacological agents to modulate pathologically high nitric oxide (NO) levels in inflammation, sepsis, and stroke. In this article, we discuss the approach for targeting the unique (6R)-5,6,7,8-tetrahydro-L-biopterin (H4Bip) binding site of NOS by appropriate inhibitors. This binding site maximally increases enzyme activity and NO production from the substrate L-arginine upon cofactor binding. The first generation of H4Bip-based NOS inhibitors was based on 4-amino H4Bip derivatives in analogy to anti-folates such as methotrexate. In addition, we discuss the structure-activity relationship of a related series of 4-oxo-pteridine derivatives. Furthermore, molecular modeling studies provide an understanding of pterin antagonism on a structural level based on favorable and unfavorable interactions between protein binding site and ligands. These techniques include 3D-QSAR (CoMFA, CoMSIA) to understand ligand affinity and GRID/consensus principal component analysis (CPCA) to learn about selectivity requirements. Collectively these approaches, in combination with the presented SAR and structural data, provide useful information for the design of novel NOS inhibitors with increased isoform selectivity.

Animals↗

PubMed2005

Pre- and postnatal diagnosis of tyrosine hydroxylase deficiency.

OBJECTIVES: Tyrosine hydroxylase (TH) is a key enzyme in the biosynthesis of dopamine, epinephrine and norepinephrine. The primary diagnosis of TH deficiency is based on the measurement of neurotransmitter metabolites and pterins in the cerebrospinal fluid, and the final diagnosis is made by detection of mutations in the TH gene. The clinical expression varies with presentations as infantile parkinsonism, L-dopa responsive spastic paraplegia, or as a progressive severe encephalopathy. Treatment with L-dopa is not always sufficient and a number of patients with poor or no response to L-dopa have recently been described. METHODS: TH is not expressed in amniotic fluid cells or in chorionic villus, so prenatal diagnosis by measurement of the enzyme activity is not possible. The only possibility of a prenatal diagnosis is by analyzing the TH gene for mutations. RESULTS: Here we describe a case of severe TH deficiency, identification of two novel mutations (p.R328W and p.T399M) and most importantly, the first prenatal diagnosis of this disease. CONCLUSIONS: The availability of prenatal diagnosis offers the parents new options. They may use the result as preparation for the birth of a child with TH deficiency, or they may decide termination of an affected pregnancy.

Brain Diseases, Metabolic, Inborn↗

PubMed2001

Evaluation of a fetus at risk for dihydropteridine reductase deficiency by direct mutation analysis using denaturing gradient gel electrophoresis.

Dihydropteridine reductase (DHPR) is an enzyme involved in the recycling of tetrahydrobiopterin (BH(4)), which is an obligate co-factor of the aromatic amino acid hydroxylases. DHPR deficiency is a rare, autosomal recessive disorder caused by mutations in the QDPR gene. DHPR-deficient patients are diagnosed by a lack of response to a low phenylalanine diet and by severe neurological symptoms. Final diagnosis is made by measurements of neurotransmitters and pterin metabolites in cerebrospinal fluid (CSF) and urine, in addition to DHPR enzyme activity, which can be assessed in whole red blood cells. Treatment of DHPR deficiency can be difficult and the outcome is not always satisfying, even if all treatment strategies are followed. Therefore prenatal diagnosis is of great importance in affected families. Prenatal diagnosis is possible by measuring DHPR activity in different cell types but this is time consuming. More than 25 different mutations have to date been identified in the QDPR gene and direct identification of a mutation in a fetus would be easy and rapid. We have developed a method based on denaturing gradient gel electrophoresis (DGGE) for the analysis of the QDPR gene. The method is useful for rapid and simultaneous scanning of all exons and flanking intronic sequences of the QDPR gene. We describe the first prenatal diagnosis conducted using this method.

Alleles↗

PubMed1995

Deletion mutants of tyrosine hydroxylase identify a region critical for heparin binding.

Phenylalanine hydroxylase, tyrosine hydroxylase, and tryptophan hydroxylase constitute a family of tetrahydropterin-dependent aromatic amino acid hydroxylases. It has been proposed that each hydroxylase is composed of a conserved C-terminal catalytic domain and an unrelated N-terminal regulatory domain. Of the three, only tyrosine hydroxylase is activated by heparin and binds to heparin-Sepharose. A series of N-terminal deletion mutants of tyrosine hydroxylase has been expressed in Escherichia coli to identify the heparin-binding site. The mutants lacking the first 32 or 68 amino acids bind to heparin-Sepharose. The mutant lacking 76 amino acids binds somewhat to heparin-Sepharose and the proteins lacking 88 or 128 do not bind at all. Therefore, an important segment of the heparin-binding site must be composed of the region from residues 76 to 90. All of the deletion mutants are active, and the Michaelis constants for pterins and tyrosine are similar among all the mutant and wild-type enzymes.

Amino Acid Sequence↗

PubMed1995

Molecular cloning and expression of biotin sulfoxide reductase from Rhodobacter sphaeroides forma sp. denitrificans.

Biotin sulfoxide reductase catalyzes the conversion of d-biotin d-sulfoxide (BSO) to d-biotin. Oligonucleotides directed against common sequences in Escherichia coli biotin sulfoxide reductase and in Rhodobacter sphaeroides f.sp. denitrificans dimethyl sulfoxide reductase have been utilized to amplify by PCR a 651-bp fragment from R. sphaeroides total genomic DNA that showed a high degree of sequence similarity with both E. coli biotin sulfoxide reductase and R. sphaeroides dimethyl sulfoxide reductase. Screening of a genomic cosmid library, prepared from R. sphaeroides genomic DNA, with this probe resulted in the isolation of a 7-kb EcoRI-EcoRI fragment that contained the complete coding region for R. sphaeroides BSO reductase which has been sequenced. The sequence data indicated a single open reading frame of 2231 nucleotides encoding a protein of 744 amino acid residues corresponding to a subunit molecular weight of 80,234 Da. The translated protein sequence contained the prokaryotic Mo-pterin signatures 2 and 3 (Mo-cofactor binding motifs) and a ATP/GTP-binding P-loop. The R. sphaeroides BSO reductase sequence showed 51% sequence similarity with the corresponding E. coli enzyme. In addition, there were only two conserved cysteines between the two BSO reductase sequences. The R. sphaeroides gene was demonstrated, by complementation, to rescue a mutant E. coli strain that was deficient in BSO reductase when grown on BSO as the sole source of biotin. When expressed from the FLAG*Shift 12c expression vector, in the presence of IPTG, the BSO reductase gene encoded a protein of approximately 80 kDa, which cross-reacted with the anti-FLAG monoclonal antibody and exhibited BSO reductase activity by the disk microbiological assay.

Amino Acid Sequence↗

PubMed1998

Xanthine oxidase reaction with nitric oxide and peroxynitrite.

Nitric oxide (.NO) and peroxynitrite (ONOO-) inhibit enzymes that depend on metal cofactors or oxidizable amino acids for activity. Since xanthine oxidase (XO) is a 2(2Fe2S) enzyme having essential sulfhydryl groups linked with Mo-pterin cofactor function, the influence of .NO and ONOO- on purified bovine XO was determined. Physiological (</=1 microM) and supraphysiological (</=100 microM) concentrations of dissolved .NO gas did not inhibit the catalytic activity or alter the spectral characteristics of XO at 25 degreesC and pH 7.0, differing from reports showing XO inhibition by .NO. The apparent decrease in XO activity observed previously was the result of depressed rates of uric acid accumulation in XO assay systems, due to ONOO--mediated oxidation of uric acid upon reaction of residual .NO with XO-derived superoxide (O*-2). Nitric oxide derived from S-nitrosoglutathione also did not inhibit cultured vascular endothelial cell XO activity. In contrast, purified and vascular endothelial cell catalase, a heme enzyme reversibly inhibited by .NO, was inhibited by similar concentrations and rates of production of . NO. In contrast to .NO, ONOO- inhibited XO (0.2 microM, 50 mU/ml) with an IC50 of 57 microM (for 3 microM/min infusion of ONOO-) or 120 microM (for bolus addition of ONOO-). Addition of 1% bovine serum albumin, 50 microM xanthine, or 10 microM uric acid protected XO from inactivation by ONOO-. Thus, in the presence of purine substrates and other more readily oxidized components of the biological milieu, XO should not be inhibited by either .NO or ONOO-. These observations reveal that .NO will not serve as an indirect antioxidant by inhibiting XO-derived production of reactive species and that the XO-derived products O*-2 and uric acid readily modify the reactivities of .NO and ONOO-.

Animals↗

PubMed1999

Mutant PTR1 proteins from Leishmania tarentolae: comparative kinetic properties and active-site labeling.

PTR1, the gene promoting MTX resistance following gene amplification or DNA transfection in Leishmania tarentolae and selected mutants, has been cloned and heavily overexpressed (>100 mg/liter) in Escherichia coli strain BL21 (DE3). Protein has been purified, essentially to homogeneity, in two steps, via ammonium sulfate precipitation and chromatography on DEAE-Trisacryl. The active proteins are tetramers and display optimal pteridine reductase activity at pH 6.0 using biopterin as substrate and NADPH as the reduced dinucleotide cofactor. 2,4-Diaminopteridine substrate analogues are strong competitive inhibitors (K(i) approximately 38 --> 3 nM) against the pterin substrate and both NADP(+) and folate are inhibitors although somewhat weaker. Dihydropteridines are poor substrates compared to the fully oxidized pteridine. Kinetic analysis affords the usual Michaelis constants and in addition shows that inhibition by NADP(+) allows the formation of ternary nonproductive complexes with folate. The kinetic results are consistent with a sequential ordered bi-bi kinetic mechanism in which first NADPH and then pteridine bind to the free enzyme. Sequence comparisons suggest that PTR1 belongs to the short-chain dehydrogenase/reductase (SDR) family containing an amino-terminal glycine-rich dinucleotide binding site plus a catalytic Y(Xaa)(3)K motif. In accord with this observation, the mutants K16A, Y37D, and R39A and the double mutants K17A:R39A and Y37D:R39A all show a two- to threefold lower binding affinity for NADPH and exhibit low or zero activity. Two Y(Xaa)(3)K regions are present in wild-type PTR1 at 152 and 194. Only Y194F gives protein with zero activity. This observation coupled with affinity labeling of PTR1 by oNADP(+) (2', 3'-dialdehyde derivative of NADP(+)) followed by NaBH(4) reduction, V8 protease digestion, and mass spectral analysis suggests that the motif participating in catalysis is that at 194. The mutation K198Q eliminates inactivation by oNADP(+) supporting the hypothesis that K198 is associated with nucleotide orientation, as has been demonstrated for similar lysine residues in other members of the SDR family.

Animals↗

PubMed2001

Assimilatory nitrate reductase: lysine 741 participates in pyridine nucleotide binding via charge complementarity.

Assimilatory NADH:nitrate reductase (EC 1.6.6.1), a complex Mo-pterin-, cytochrome b557-, and FAD-containing protein, catalyzes the regulated and rate-limiting step in the utilization of inorganic nitrogen by high plants. With a recombinant, histidine-tagged form of the spinach nitrate reductase flavin domain, site-directed mutagenesis has been utilized to examine the role of lysine 741 in binding the reducing substrate, NADH. Seven individual mutants, corresponding to K741R, K741H, K741A, K741E, K741M, K741Q, and K741P, have been engineered and six of the resulting proteins purified to homogeneity. With the exception of K741P, all the mutants were obtained as functional flavoproteins which retained FAD as the sole prosthetic group and exhibited spectroscopic properties comparable to those of the wild-type domain, indicating that the amino acid substitutions had no effect on FAD binding. In contrast, all the mutants were found to have altered NADH:ferricyanide reductase (NADH:FR) activity with mutations affecting both kcat and K(NADH)m, which decreased and increased, respectively. At pH 7.0, kcat decreased in the order WT > K741R > K741A > K741H > K741E > K741M > K741Q while K(NADH)m increased in the same order. The most efficient mutant, K741R, retained 80% of the wild-type NADH:FR activity, while in contrast the most inefficient mutant, K741Q, retained only 18% of the wild-type NADH:FR activity together with a 118-fold increased K(NADH)m. pH studies of K741H revealed that both kcat and K(NADH)m were pH-dependent, with enhanced activity observed at acidic pH. These results indicated that retention of a positively charged side chain at position 741 in the spinach nitrate reductase primary sequence is important for the efficient binding and subsequent oxidation of NADH and that the positively charged side chain enhances nucleotide binding via charge complementarity with the negatively charged pyrophosphate moiety.

Amino Acid Sequence↗

PubMed1994

Molecular cloning, tissue expression of human xanthine dehydrogenase.

Xanthine dehydrogenase (XDH, EC 1.1.1.204) is a molybdenum iron-sulphur flavin hydroxylase which oxidizes a variety of purines, pterins and other heterogenic nitrogen compounds, serving as a rate-limiting enzyme in nucleic acid degradation. In this work, we have isolated and sequenced cDNA clones of human liver XDH. The obtained cDNA covers 4577 bases of human liver XDH mRNA with a 63 bp 5'-end untranslated region and a 515 bp 3'-end untranslated region. A termination codon TGA and a polyadenylation signal AATAAA were identified. An open reading frame encodes 1333 amino acid residues. The assignment of the N-terminal was confirmed by directly sequencing that region of purified human milk XDH. Northern blot analysis shows that the human XDH gene is widely expressed in human tissues.

Amino Acid Sequence↗