Mutation to phenylalanine of tyrosine 371 in tyrosine hydroxylase increases the affinity for phenylalanine

The aromatic amino acid hydroxylases tyrosine and phenylalanine hydroxylase both contain non-heme iron, utilize oxygen and tetrahydrobiopterin, and are tetramers of identical subunits. The catalytic domains of these enzymes are homologous, and recent X-ray crystallographic analyses show the active sites of the two enzymes are very similar. The hydroxyl oxygens of tyrosine 371 in tyrosine hydroxylase and of tyrosine 325 of phenylalanine hydroxylase are 5 and 4.5 A, respectively, away from the active site iron in the enzymes. To determine whether this residue has a role in the catalytic mechanism as previously suggested [Erlandsen, H., et al. (1997) Nat. Struct. Biol. 4, 995-1000], tyrosine 371 of tyrosine hydroxylase was altered to phenylalanine by site-directed mutagenesis. The Y371F protein was fully active in tyrosine hydroxylation, eliminating an essential mechanistic role for this residue. There was no change in the product distribution seen with phenylalanine or 4-methylphenylalanine as a substrate, suggesting that the reactivity of the hydroxylating intermediate was unaffected. However, the KM value for phenylalanine was decreased 10-fold in the mutant protein. These results are interpreted as an indication of greater conformational flexibility in the active site of the mutant protein.     

Restriction map polymorphism in the forked and vermilion regions of Drosophila melanogaster

Restriction map polymorphism at two X linked foci, forked and vermilion of Drosophila melanogaster was studied in three natural populations. The estimates of nucleotide variation were theta = 0.003 and pi = 0.002 for the forked region and theta = 0.004 and pi = 0.002 for the vermilion region. Three insertions (> 500 bp) were observed at each locus. Typical of other regions of this species each of these large insertions was unique in the sample. Non-random association among polymorphisms was common at the vermilion locus, while the forked locus was not polymorphic enough to test linkage disequilibrium. The amounts of restriction site and size variation in the vermilion and forked were within the range observed for other loci of D. melanogaster.     

Structure of tetrameric human phenylalanine hydroxylase and its implications for phenylketonuria

Phenylalanine hydroxylase (PheOH) catalyzes the conversion of L-phenylalanine to L-tyrosine, the rate-limiting step in the oxidative degradation of phenylalanine. Mutations in the human PheOH gene cause phenylketonuria, a common autosomal recessive metabolic disorder that in untreated patients often results in varying degrees of mental retardation. We have determined the crystal structure of human PheOH (residues 118-452). The enzyme crystallizes as a tetramer with each monomer consisting of a catalytic and a tetramerization domain. The tetramerization domain is characterized by the presence of a domain swapping arm that interacts with the other monomers forming an antiparallel coiled-coil. The structure is the first report of a tetrameric PheOH and displays an overall architecture similar to that of the functionally related tyrosine hydroxylase. In contrast to the tyrosine hydroxylase tetramer structure, a very pronounced asymmetry is observed in the phenylalanine hydroxylase, caused by the occurrence of two alternate conformations in the hinge region that leads to the coiled-coil helix. Examination of the mutations causing PKU shows that some of the most frequent mutations are located at the interface of the catalytic and tetramerization domains. Their effects on the structural and cellular stability of the enzyme are discussed.     

Phenylalanine hydroxylase from Chromobacterium violaceum. Uncoupled oxidation of tetrahydropterin and the role of iron in hyroxylation

A gene encoding phenylalanine hydroxylase has been cloned from Chromobacterium violaceum and expressed in Escherichia coli. The purified phenylalanine hydroxylase contains copper, which does not support enzymatic activity. Upon removal of copper by dithiothreitol (DTT), the enzyme contains substoichiometric amounts of calcium and zinc but little or no redox-active metal ions. The copper-depleted hydroxylase catalyzes the phenylalanine-dependent oxidation of 6, 7-dimethyltetrahydropterin (DMPH4) by O2 in a reaction in which phenylalanine is not hydroxylated and does not appear to undergo a chemical change, and hydrogen peroxide is produced. Analogs of phenylalanine also activate the oxidation of DMPH4. Both the copper-phenylalanine hydroxylase and the copper-depleted hydroxylase catalyze the hydroxylation of phenylalanine in the presence of DTT and FeSO4 in a reaction in which hydrogen peroxide is not produced. The apparent values of Km for Fe2+ and DTT are 0.28 microM and 1.1 mM, respectively, at 1.0 mM phenylalanine, 120 microM DMPH4 and pH 7. 4 and 23 degreesC. The apparent value of kcat is 14.3 s-1 under these conditions. Glutathione, mercaptoethanol, and dihydrolipoate support the hydroxylation of phenylalanine essentially as well as DTT. Incubation of copper-depleted hydroxylase with FeSO4, phenylalanine, and DTT followed by gel permeation chromatography leads to an iron-hydroxylase containing approximately 1 molecule of iron per molecule of enzyme. The iron-hydroxylase displays an optical absorption band extending from 300 to 600 nm, and it catalyzes the hydroxylation of phenylalanine at the same maximum rate as the iron-activated hydroxylase but does not require added Fe2+. We conclude that iron participates in the hydroxylation of phenylalanine. Iron is not required for the oxidation of DMPH4, although it may exert a modest acceleration effect. A hypothetical mechanism is presented wherein the reaction of iron with the putative 4a-hydroperoxy-DMPH4 leads to 4a-hydroxy-DMPH4 and a high valent iron-oxy species. The iron-oxy species is postulated to react with phenylalanine in the hydroxylation process.     

Reliability of potential clinical measures of muscle tone in the elbows of patients after stroke

In order to establish a genotype-phenotype relationship, we have identified both mutant phenylalanine hydroxylase (PAH) genes in 108 phenylketonuria (PKU) patients (27 different alleles, 54 different genotypes). One major group of patients with very high pretreatment phenylalanine values ("classical" PKU) exclusively comprised homozygotes of the PKU mutations I65T, G272X, F299C, Y356X, R408W, IVS12nt1, and compound heterozygotes of various combinations of these alleles with G46S, R261Q, R252W, A259T, R158Q, D143G, R243X, E280K, or Y204C. A second major group of patients with lower phenylalanine values ("mild" PKU) comprised mutations A300S, R408Q, Y414C in various compound heterozygous states, and R261Q, R408Q, Y414C in homozygotes. The phenylalanine values in these groups were non-overlapping. In addition, a smaller group of patients formed the transition between the two main groups. In sib pairs 4 of 15 had discordant pretreatment phenylalanine values. Conclusion: Our results are consistent with the view that allelic heterogeneity at the PAH locus dominates the biochemical phenotype in PKU and that genotype information is able to predict the metabolic phenotype in PKU patients.     

A physical comparison of chromosome III in six strains of Saccharomyces cerevisiae

We have tested the clones used in the European Yeast Chromosome III Sequencing Programme for possible artefacts that might have been introduced during cloning or passage through Escherichia coli. Southern analysis was performed to compare the BamHI, EcoRI, HindIII and PstI restriction pattern for each clone with that of the corresponding locus on chromosome III in the parental yeast strain. In addition, further enzymes were used to compare the restriction maps of most clones with the map predicted by the nucleotide sequence (Oliver et al., 1992). Only four of 506 6-bp restriction sites predicted by the sequence were not observed experimentally. No significant cloning artefacts appear to disrupt the published sequence of chromosome III. The restriction patterns of six yeast strains have also been compared. In addition to two previously identified sites of Ty integration on chromosome III (Warmington et al., 1986; Stucka et al., 1989; Newlon et al., 1991), a new polymorphic site involving Ty retrotransposition (the Far Right-Arm transposition Hot-Spot, FRAHS) has been identified close to CRY1. On the basis of simple restriction polymorphisms, the strains S288C, AB972 and W303-1b are closely related, while XJ24-24a and J178 are more distant relatives of S288C. A polyploid distillery yeast is heterozygous for many polymorphisms, particularly on the right arm of the chromosome.     

Polymorphisms of the calcitonin receptor gene are associated with bone mineral density in postmenopausal Italian women

Recognition of a major genetic component in bone mass determination represented the basis for studies aiming to the identification of underlying major and minor genes. Bone mineral density (BMD) represents the continuous trait to be quantified in order to evaluate segregation of candidate genes with risk of osteoporosis. Polymorphisms at the vitamin D receptor (VDR), estrogen receptor, (ER), collagen type I, and interleukin 6 (IL6) gene loci have been correlated to BMD. However, in a polygenic disorder, such as osteoporosis, the number of genes expected to influence BMD is very large. In the present study we examined the presence of restriction fragment length polymorphisms (RFLPs) for the calcitonin receptor (CTR) gene in postmenopausal women. We identified a polymorphic (Tt) site at the CTR gene locus using the Taq I restriction fragment enzyme. Three genotypes were observed, whose Tt was the most frequent in our population (49.7%). In addition, Ancova analysis and Tukey's test showed that women with tt genotype had significantly lower lumbar BMD in comparison with Tt genotype (Tukey's test: p = 0.005). In conclusion, evidence of RFLPs at the CTR gene locus in Caucasian postmenopausal women of Italian origin made it possible to identify the involvement of another gene, the CTR gene, in the determination of bone mass.     

Mucopolysaccharidosis IVA: four new exonic mutations in patients with N-acetylgalactosamine-6-sulfate sulfatase deficiency

We report four new mutations in Japanese patients with mucopolysaccharidosis IVA (MPSIVA) who were heterozygous for a common double gene deletion. A nonsense mutation of CAG to TAG at codon 148 in exon 4 was identified, resulting in a change of Q to a stop codon and three missense mutations. V (GTC) to A (GCC) at codon 138 in exon 4, P (CCC) to S (TCC) at codon 151 in exon 5, and P (CCC) to L (CTC) at codon 151 in exon 5. Introduction of these mutations into the normal GALNS cDNA and transient expression in cultured fibroblasts resulted in a significant decrease in the enzyme activity. V138A and Q148X mutations result in changes of restriction site, which were analyzed by restriction-enzyme assay. P151S and P151L mutations that did not alter the restriction site were detected by direct sequencing or allele specific oligohybridization. Detection of the double gene deletion was initially done using Southern blots and was confirmed by PCR. Haplotypes were determined using seven polymorphisms to the GALNS locus in families with the double gene deletion. Haplotype analysis showed that the common double gene deletion occurred on a single haplotype, except for some variation in a VNTR-like polymorphism. This finding is consistent with a common founder for all individuals with this mutation.     

A new restriction fragment length polymorphism from Cryptosporidium parvum identifies genetically heterogeneous parasite populations and genotypic changes following transmission from bovine to human hosts

Length and restriction site polymorphism within a 2.8-kb threonine-rich open reading frame from Cryptosporidium parvum was identified and used to determine the genotypes of isolates from calves and humans. In agreement with observations of other genetic loci, all calf isolates were identical at this locus. In contrast, human isolates showed two profiles, one found exclusively in humans and one a superposition of both profiles, which were indicative of heterogeneous parasite populations. PCR fingerprints were consistent with a change in the genetic profile of C. parvum isolates following transmission from bovine to human hosts.     

The influence of positive selection on RAG expression in thymocytes

Many human cancers present deletions of the short arm of chromosome 17, which includes the TP53 locus. We detected a new polymorphism in intron 2 of the TP53 gene using PCR-SSCP and used this polymorphic site as a marker to detect loss of heterozygosity in 135 human tumors (73 soft tissue sarcomas, and 48 colorectal and 14 bladder carcinomas). Heterozygosity for this site was 41.5% in this study group and tumor-specific loss of alleles occurred in 43% of informative cases. Allelic losses were more frequently detected at this site than at that in which restriction fragment length polymorphism (RFLP) is located, as detected by the pHp53B probe. It is concluded that this novel approach has several advantages, including detection of a high incidence of informative cases and minimal tissue requirements.     

Human pterin-4 alpha-carbinolamine dehydratase/dimerization cofactor of hepatocyte nuclear factor-1 alpha. Characterization and kinetic analysis of wild-type and mutant enzymes

Pterin-4a-carbinolamine dehydratase/dimerization cofactor for hepatocyte nuclear factor-1 alpha is a protein with two different functions. We have overexpressed and purified the human wild-type protein, and its Cys81Ser and Cys81Arg mutants. The Cys81Arg mutant has been proposed to be causative in a hyperphenylalaninaemic patient [Citron, B. A., Kaufman, S., Milstien, S., Naylor, E. W., Greene, C. L. & Davis, M. D. (1993) Am. J. Hum. Genet. 53, 768-774]. The dehydratase behaves as a tetramer on gel filtration, while cross-linking experiments showed mono-, di-, tri-, and tetrameric forms, irrespective of the presence of the single Cys81. Sulfhydryl-modifying reagents did not affect the activity, but rather showed that Cys81 is exposed. Various pterins bind and quench the tryptophan fluorescence suggesting the presence of a specific binding site. The fluorescence is destroyed upon light irradiation. Wild-type and the Cys81Ser protein enhance the rate of the phenylalanine hydroxylase assay approximately 10-fold, a value similar to that of native dehydratase from rat liver; the Cys81Arg mutant, in contrast, has significantly lower activity. This is compatible with the hypothesis that the dehydratase is a rate-limiting factor for the in vivo phenylalanine hydroxylase reaction. The three proteins enhance the spontaneous dehydration of the synthetic substrate 6,6-dimethyl-7,8-dihydropterin-4a-carbinolamine approximately 50-70-fold at 4 degrees C and pH 8.5. The results are discussed in view of the recently solved three-dimensional structure of the enzyme [Ficner, R., Sauer, U. W., Stier, G. & Suck, D. (1995) EMBO J. 14, 2032-2042].     

DNA polymorphisms in the ACE gene, serum ACE activity and the risk of nephropathy in insulin-dependent diabetes mellitus

BACKGROUND: To determine the relationship between DNA polymorphisms in the angiotensin I converting enzyme (ACE) gene, serum ACE activity and the risk of diabetic nephropathy. METHODS: A case-control study was carried out in a population of Jewish insulin-dependent diabetes mellitus (IDDM) patients. Cases (77 IDDM patients with diabetic nephropathy) and controls (89 IDDM patients with normoalbuminuria) were genotyped with PCR protocols for detecting two DNA polymorphisms in the ACE gene: one in intron 7 detected with the restriction enzyme PstI and the other in intron 16 identified as an insertion/deletion (I/D). RESULTS: The risk of nephropathy was increased only in patients homozygous for the allele with the PstI site. These homozygotes had a nephropathy risk that was 2.3 times (95% C.I.: 1.2-4.5) that of the other genotypes. Furthermore, these individuals did not have elevated serum ACE activity. CONCLUSIONS: The results of this study are evidence that the risk of diabetic nephropathy in IDDM is influenced by genetic variability at the ACE locus, but the responsible variant is not the I/D polymorphism in intron 16. Our findings require further studies in other populations.     

Crystallization and preliminary diffraction analysis of a truncated homodimer of human phenylalanine hydroxylase

A recombinant truncated form (delta1-102/delta428-452) of the non-heme iron-dependent metalloenzyme human phenylalanine hydroxylase (hPAH, phenylalanine 4-monooxygenase; EC 1.14.16.1) was expressed in E. coli, purified to homogeneity as a homodimer (70 kDa) and crystallized using the hanging drop vapour diffusion method. The crystals are orthorhombic, space group C222 with cell dimensions of a = 66.6 A, b = 108.4 A, c = 125.7 A. The calculated packing parameter (Vm) is 3.24 A3/Da with four 2-fold symmetric dimers (or eight momomers) in the unit cell. Data have been collected to 2.0 A resolution.     

Differential effects of mutations in substrate recognition site 1 of cytochrome P450 2C2 on lauric acid and progesterone hydroxylation

Mutations at amino acid positions 107-120, which are part of a predicted substrate recognition site [Gotoh, O. (1992) J. Biol. Chem. 267, 83-90], were analyzed in C2MstC1, a chimera of P450 2C2 and P450 2C1. This hybrid protein has a new activity for progesterone C21-hydroxylation in addition to the lauric acid (omega-1)hydroxylase activity present in both parent proteins. Various substitutions for highly conserved glycines at positions 111 and 117 and tryptophan at position 120 strongly decreased the lauric acid hydroxylase activity of P450 2C2 and C2MstC1 and the progesterone hydroxylase activity of C2MstC1. Activities of mutant proteins with substitutions at 107, 108, and 112-115 were also strongly reduced. Modest or no decreases in activity were observed for substitutions at 109, 110, 116, 118, and 119. Lauric acid hydroxylase activity decreased more in most C2MstC1 mutants than in those of P450 2C2, particularly at positions 107 and 108. A substitution of phenylalanine for valine-112 reduced progesterone hydroxylation by 30-fold while only moderately reducing lauric acid hydroxylase by 40%. This differential effect on two dissimilar substrates demonstrates the importance of residue 112 for substrate interactions. The results are consistent with a model in which residues 107-110 align with the B'-helix of the bacterial proteins P450cam and P450BM-3. This helix is followed by a substrate-contacting loop from 111 to 116, and residues 117-120 align with the C-helices of the bacterial proteins In this alignment, Trp-120 is positioned behind the heme such that it could participate in electron transfer from reductase.(ABSTRACT TRUNCATED AT 250 WORDS)     

Crystal structure of tyrosine hydroxylase at 2.3 A and its implications for inherited neurodegenerative diseases

Tyrosine hydroxylase (TyrOH) catalyzes the conversion of tyrosine to L-DOPA, the rate-limiting step in the biosynthesis of the catecholamines dopamine, adrenaline, and noradrenaline. TyrOH is highly homologous in terms of both protein sequence and catalytic mechanism to phenylalanine hydroxylase (PheOH) and tryptophan hydroxylase (TrpOH). The crystal structure of the catalytic and tetramerization domains of TyrOH reveals a novel alpha-helical basket holding the catalytic iron and a 40 A long anti-parallel coiled coil which forms the core of the tetramer. The catalytic iron is located 10 A below the enzyme surface in a 17 A deep active site pocket and is coordinated by the conserved residues His 331, His 336 and Glu 376. The structure provides a rationale for the effect of point mutations in TyrOH that cause L-DOPA responsive parkinsonism and Segawa's syndrome. The location of 112 different point mutations in PheOH that lead to phenylketonuria (PKU) are predicted based on the TyrOH structure.     

Long-term effect of RU24722 on tyrosine hydroxylase in the rat locus coeruleus: differential effects of two enantiomeric forms

RU24722, as a racemic mixture, has been found to act on neuronal activity and the long-term regulation of tyrosine hydroxylase in the locus coeruleus of the rat. In this study, the effects of two enantiomeric derivatives of RU24722 (3 alpha and 16 alpha forms), as compared to the racemic form itself, are studied. The short-term effect was estimated 20 min after treatment by measuring variations in 3,4-dihydroxyphenylacetic acid content in the locus coeruleus. The long-term effect was determined by evaluating tyrosine hydroxylase protein concentration in the locus coeruleus 3 days after a single injection. Comparison of actions of both enantiomers showed that the 16 alpha form was 3-fold more potent in eliciting tyrosine hydroxylase protein elevations at three days, whereas the 3 alpha isomer increased 3,4-dihydroxyphenylacetic acid content 2-fold more in the short-term. These results seem to show that the 16 alpha configuration is crucial for the long term regulation of tyrosine hydroxylase protein elicited by RU24722 within the locus coeruleus.     

The porcine TTR locus maps to chromosome 6q

The sequence of a cDNA clone encoding porcine transthyretin (prealbumin) was used to develop polymorphic markers for the TTR locus. The single-strand conformation polymorphism (SSCP) detected is caused by a silent A/T mutation in the penultimate coding codon and can also be revealed as a SacI restriction fragment length polymorphism (RFLP). The TTR locus was mapped to chromosome 6q by segregation and linkage analysis with these polymorphisms. This assignment confirms the predictions of homology between human chromosome 18 and pig chromosome 6q2.5-q2.6.     

Alpha-methylphenylalanine, a new inducer of chronic hyperphenylalaninemia in sucling rats

alpha-Methylphenylalanine reduces the phenylalanine hydroxylase activity of rat liver by 75 percent. Daily injections of this substance (plus phenylalanine) into rats from the 3rd to 15th day of age had no obvious toxic effects, and maintained a plasma concentration of phenylalanine comparable to that of phenylketonuric subjects.