Aromatic Amino Acid Hydroxylases Research Articles

Elucidation of post‐translational mechanisms for regulation of GTP cyclohydrolase I

GTP cyclohydrolase I (GTPCH) is the first and rate-limiting enzyme in the tetrahydrobiopterin (BH4) biosynthetic pathway. BH4 is an obligate cofactor for the nitric oxide (NO) synthases and the aromatic amino acid hydroxylases. GTPCH feedback regulatory protein (GFRP) mediates feedback inhibition of GTPCH by BH4, as well as stimulation of GTPCH by phenylalanine. We hypothesized that additional proteins associate with the GTPCH-GFRP complex and contribute to subcellular localization and/or enzymatic regulation of GTPCH-GFRP. Additionally, evidence suggests that GTPCH-GFRP is subject to regulation by phosphorylation, although relevant sites of in vivo modification remain largely unspecified. To identify binding proteins and phosphorylation sites involved in the regulation of GTPCH-GFRP, we developed affinity capture approaches for gentle purification of endogenous GTPCH-GFRP complexes. Capture by His6-tagged GFRP was successful in enriching GTPCH to levels adequate for identification by mass spectrometry. Comparisons of different incubation conditions confirmed that substrate, effectors and inhibitors of GTPCH activity can all increase levels of GTPCH bound to GFRP. More efficient enrichment of GTPCH is being accomplished using a tandem affinity purification (TAP) procedure. The TAP system’s dual elution steps are highly specific and eliminate the need for harsh washing that could disrupt protein complexes. Nanoflow liquid chromatography-coupled tandem mass spectrometry (nLC/MS/MS) is being used to identify co-purifying protein binding partners of GTPCH-GFRP and to reveal post-translational modification sites.

Interaction of human GTP cyclohydrolase I with its splice variants

Tetrahydrobiopterin is an essential cofactor for aromatic amino acid hydroxylases, ether lipid oxidase and nitric oxide synthases. Its biosynthesis in mammals is regulated by the activity of the homodecameric enzyme GCH (GTP cyclohydrolase I; EC 3.5.4.16). In previous work, catalytically inactive human GCH splice variants differing from the wild-type enzyme within the last 20 C-terminal amino acids were identified. In the present study, we searched for a possible role of these splice variants. Gel filtration profiles of purified recombinant proteins showed that variant GCHs form high-molecular-mass oligomers similar to the wild-type enzyme. Co-expression of splice variants together with wild-type GCH in mammalian cells revealed that GCH levels were reduced in the presence of splice variants. Commensurate with these findings, the GCH activity obtained for wild-type enzyme was reduced 2.5-fold through co-expression with GCH splice variants. Western blots of native gels suggest that splice variants form decamers despite C-terminal truncation. Therefore one possible explanation for the effect of GCH splice variants could be that inactive variants are incorporated into GCH heterodecamers, decreasing the enzyme stability and activity.

Functional Domains of Human Tryptophan Hydroxylase 2 (hTPH2)

Tryptophan hydroxylase (TPH) is the rate-limiting enzyme in serotonin biosynthesis. A novel gene, termed TPH2, has recently been described. This gene is preferentially expressed in the central nervous system, while the original TPH1 is the peripheral gene. We have expressed human tryptophan hydroxylase 2 (hTPH2) and two deletion mutants (NDelta150 and NDelta150/CDelta24) using isopropyl beta-D-thiogalactopyranoside-free autoinduction in Escherichia coli. This expression system produced active wild type TPH2 with relatively low solubility. The solubility was increased for mutants lacking the NH(2)-terminal regulatory domain. The solubility of hTPH2, NDelta150, and NDelta150/CDelta24 are 6.9, 62, and 97.5%, respectively. Removal of the regulatory domain also produced a more than 6-fold increase in enzyme stability (t((1/2)) at 37 degrees C). The wild type hTPH2, like other members of the aromatic amino acid hydroxylase superfamily, exists as a homotetramer (236 kDa on size exclusion chromatography). Similarly, NDelta150 also migrates as a tetramer (168 kDa). In contrast, removal of the NH(2)-terminal domain and the COOH-terminal, putative leucine zipper tetramerization domain produces monomeric enzyme (39 kDa). Interestingly, removal of the NH(2)-terminal regulatory domain did not affect the Michaelis constants for either substrate but did increase V(max) values. These data identify the NH(2)-terminal regulatory domain as the source of hTPH2 instability and reduced solubility.

Tetrahydrobiopterin and Cardiovascular Disease

Tetrahydrobiopterin (BH4) is an essential cofactor for the aromatic amino acid hydroxylases, which are essential in the formation of neurotransmitters, and for nitric oxide synthase. It is presently used clinically to treat some forms of phenylketonuria (PKU) that can be ameliorated by BH4 supplementation. Recent evidence supports potential cardiovascular benefits from BH4 replacement for the treatment of hypertension, ischemia-reperfusion injury, and cardiac hypertrophy with chamber remodeling. Such disorders exhibit BH4 depletion because of its oxidation and/or reduced synthesis, which can result in functional uncoupling of nitric oxide synthase (NOS). Uncoupled NOS generates more oxygen free radicals and less nitric oxide, shifting the nitroso-redox balance and having adverse consequences on the cardiovascular system. While previously difficult to use as a treatment because of chemical instability and cost, newer methods to synthesize stable BH4 suggest its novel potential as a therapeutic agent. This review discusses the biochemistry, physiology, and evolving therapeutic potential of BH4 for cardiovascular disease.

Insights into the Catalytic Mechanisms of Phenylalanine and Tryptophan Hydroxylase from Kinetic Isotope Effects on Aromatic Hydroxylation

Phenylalanine hydroxylase (PheH) and tryptophan hydroxylase (TrpH) catalyze the aromatic hydroxylation of phenylalanine and tryptophan, forming tyrosine and 5-hydroxytryptophan, respectively. The reactions of PheH and TrpH have been investigated with [4-(2)H]-, [3,5-(2)H(2)]-, and (2)H(5)-phenylalanine as substrates. All (D)k(cat) values are normal with Delta117PheH, the catalytic core of rat phenylalanine hydroxylase, ranging from 1.12-1.41. In contrast, for Delta117PheH V379D, a mutant protein in which the stoichiometry between tetrahydropterin oxidation and amino acid hydroxylation is altered, the (D)k(cat) value with [4-(2)H]-phenylalanine is 0.92 but is normal with [3,5-(2)H(2)]-phenylalanine. The ratio of tetrahydropterin oxidation to amino acid hydroxylation for Delta117PheH V379D shows a similar inverse isotope effect with [4-(2)H]-phenylalanine. Intramolecular isotope effects, determined from the deuterium contents of the tyrosine formed from [4-(2)H]-and [3,5(2)H(2)]-phenylalanine, are identical for Delta117PheH and Delta117PheH V379D, suggesting that steps subsequent to oxygen addition are unaffected in the mutant protein. The inverse effects are consistent with the reaction of an activated ferryl-oxo species at the para position of the side chain of the amino acid to form a cationic intermediate. The normal effects on the (D)k(cat) value for the wild-type enzyme are attributed to an isotope effect of 5.1 on the tautomerization of a dienone intermediate to tyrosine with a rate constant 6- to7-fold that for hydroxylation. In addition, there is a slight ( approximately 34%) preference for the loss of the hydrogen originally at C4 of phenylalanine. With (2)H(5)-indole-tryptophan as a substrate for Delta117PheH, the (D)k(cat) value is 0.89, consistent with hydroxylation being rate-limiting in this case. When deuterated phenylalanines are used as substrates for TrpH, the (D)k(cat) values are within error of those for Delta117PheH V379D. Overall, these results are consistent with the aromatic amino acid hydroxylases all sharing the same chemical mechanism, but with the isotope effect for hydroxylation by PheH being masked by tautomerization of an enedione intermediate to tyrosine.

Discovery of specific tryptophan hydroxylase in the brain of the beetle Harmonia axyridis

Rabbit anti-serotonin and mouse monoclonal anti-tryptophan hydroxylase antisera were applied on the brain sections of the beetle Harmonia axyridis, butterfly Childrena zenobia, moth Antheraea pernyi and ant Camponotus japonicus, using the Streptavidin–Peroxidase immunohistochemical method and Colophony–Paraffin embedded section technique. Results revealed that all the experimental insects showed notable serotonin-like immunoreactivity in the brain. However, only the brain sections of the beetle H. axyridis were strongly labeled by mouse monoclonal anti-tryptophan hydroxylase antiserum, with the distribution pattern matching that of serotonin. These results demonstrate that specific tryptophan hydroxylase may exist in the brain of the beetle and likely reflect the diversity of serotonin synthetic mechanisms as well as the evolution of aromatic amino acid hydroxylase genes.

Structure of Chlorobium tepidum Sepiapterin Reductase Complex Reveals the Novel Substrate Binding Mode for Stereospecific Production of l-threo-Tetrahydrobiopterin

Sepiapterin reductase (SR) is involved in the last step of tetrahydrobiopterin (BH(4)) biosynthesis by reducing the di-keto group of 6-pyruvoyl tetrahydropterin. Chlorobium tepidum SR (cSR) generates a distinct BH(4) product, L-threo-BH(4) (6R-(1'S,2'S)-5,6,7,8-BH(4)), whereas animal enzymes produce L-erythro-BH(4) (6R-(1'R,2'S)-5,6,7,8-BH(4)) although it has high amino acid sequence similarities to the other animal enzymes. To elucidate the structural basis for the different reaction stereospecificities, we have determined the three-dimensional structures of cSR alone and complexed with NADP and sepiapterin at 2.1 and 1.7 A resolution, respectively. The overall folding of the cSR, the binding site for the cofactor NADP(H), and the positions of active site residues were quite similar to the mouse and the human SR. However, significant differences were found in the substrate binding region of the cSR. In comparison to the mouse SR complex, the sepiapterin in the cSR is rotated about 180 degrees around the active site and bound between two aromatic side chains of Trp-196 and Phe-99 so that its pterin ring is shifted to the opposite side, but its side chain position is not changed. The swiveled sepiapterin binding results in the conversion of the side chain configuration, exposing the opposite face for hydride transfer from NADPH. The different sepiapterin binding mode within the conserved catalytic architecture presents a novel strategy of switching the reaction stereospecificities in the same protein fold.

Intrinsic Isotope Effects on Benzylic Hydroxylation by the Aromatic Amino Acid Hydroxylases: Evidence for Hydrogen Tunneling, Coupled Motion, and Similar Reactivities

Deuterium kinetic isotope effects for hydroxylation of the methyl group of 4-methylphenylalanine have been used as a probe of the relative reactivities of the hydroxylating intermediates in the aromatic amino acid hydroxylases phenylalanine, tyrosine, and tryptophan hydroxylase. When there are three deuterium atoms in the methyl group, all three enzymes exhibit an intrinsic isotope effect of about 13. The temperature dependence of the isotope effect is consistent with moderate tunneling, with the extent of tunneling identical for all three enzymes. In the case of phenylalanine hydroxylase, the presence of the regulatory domain has no effect on the values. The intrinsic primary and secondary isotope effects were determined using 4-methylphenylalanine containing one or two deuterium atoms in the methyl group. With one deuterium atom, the intrinsic primary and secondary effects have average values of 10 and 1.1, respectively. With two deuterium atoms, the primary effects decrease to 7.4 and the secondary effect increases to 1.3, consistent with coupled motion of the primary and secondary hydrogens. The results with all three enzymes are consistent with a hydrogen abstraction mechanism. The similarities of the isotope effects and extent of tunneling establish that the reactivities of the hydroxylating intermediates in the three enzymes are essentially identical.

An oxygraphic method for determining kinetic properties and catalytic mechanism of aromatic amino acid hydroxylases

We have developed a simple and versatile oxygraphic assay procedure that can be used for determination of kinetic constants and enzyme reaction mechanisms of wild-type and mutant aromatic amino acid hydroxylases. The oxygen concentration and rate of oxygen consumption were measured continuously throughout the enzyme reaction, while aliquots of the reaction mixture were removed at regular intervals for measurement of other substrates and products. Using (6 R)-tetrahydrobiopterin as electron donor in the phenylalanine hydroxylase (PAH) reaction, a stable stoichiometry of 1:1 was obtained between the amount of oxygen consumed and the tyrosine formation. In comparison, low and variable coupling efficiency values between oxygen consumption and tyrosine formation were found using the parent unsubstituted tetrahydropterin. The application of this assay procedure to study mechanisms of disease-associated mutations was also demonstrated. Thus, the phenylketonuria-associated PAH mutant R158Q had a coupling efficiency of about 80%, compared to the wild-type enzyme under similar conditions. Furthermore, the amount of H 2O 2 produced in the reaction catalyzed by R158Q PAH was about four times higher than the amount produced by the wild-type PAH, demonstrating a possible pathogenetic mechanism of the mutant enzyme.

Possibility of the Nonenzymatic Conversion of 6-(1'-Oxo-2'-hydroxypropyl)- Tetrahydropterin to Sepiapterin

Abstract Tetrahydrobiopterin (BH4) is a cofactor for aromatic amino acid hydroxylases and nitric oxide synthase. The biosynthetic pathway of BH4 from 6-pyruvoyl-tetrahydropterin (PPH4) includes two reduction steps catalyzed by sepiapterin rcductasc (SPR). PP1I4 is reduced to 6-( 1 '-oxo-2'-hydroxypiOpyl)-tetrahydropterin (l'-OXPH4 ) or 6- ( r-hydroxy-2'-oxopropyl)-tetrahydropterin (2'-OXPH4), which is further converted to BH4. However, patients with SPR deficiency show normal urinary excretion of pterins without hyperphenylalaninemia, suggesting the existence of another BH4 biosynthetic pathway, which is not concerned with SPR in humans. Blau et al. proposed a BH4 biosynthetic salvage pathway containing nonenzymatic conversion of l'-OXPH4 to sepiapterin. In this study, the possibility of the nonenzymatic conversion of l'-OXPH4 to sepiapterin was examined by using silkworm carbonyl reductase (CR 1) and human monomeric carbonyl reductase. Since l'-OXPH4 has been suggested to be nonenzymatically converted into sepiapterin, in an incubation mixture containing PPH4 and silkworm CR I, sepiapterin was determined by its derivative as biopterin. No sepiapterin was detected in the incubation mixture when the mixture, containing 800 pmol of l'-OXPH4, was further incubated at 37°C for 2 h in darkness. This result suggests that the rate of nonenzymatic formation of sepiapterin from l'-OXPH4 is quite low. The findings obtained here indicate that the proposed pathway in which a nonenzymatic conversion of l'-OXPH4 to sepiapterin occurs may be difficult or unlikely to proceed in humans.

Allosteric mechanisms in ACT domain containing enzymes involved in amino acid metabolism

An important sequence motif identified by sequence analysis is shared by the ACT domain family, which has been found in a number of diverse proteins. Most of the proteins containing the ACT domain seem to be involved in amino acid and purine synthesis and are in many cases allosteric enzymes with complex regulation enforced by the binding of ligands. Here we explore the current understanding of the ACT domain function including its role as an allosteric module in a selected group of enzymes. We will further describe in more detail three of the proteins where some understanding is available on function and structure: i) the archetypical ACT domain protein E. coli 3PGDH, which catalyzes the first step in the biosynthesis of L-Ser, ii) the bifunctional chorismate mutase/prephenate dehydratase (P-protein) from E. coli, which catalyzes the first two steps in the biosynthesis of L-Phe, and iii) the mammalian aromatic amino acid hydroxylases, with special emphasis on phenylalanine hydroxylase, which catalyzes the first step in the catabolic degradation of L-phenylalanine (L-Phe). The ACT domain is commonly involved in the binding of a small regulatory molecule, such as the amino acids L-Ser and L-Phe in the case of 3PGDH and P-protein, respectively. On the other hand, for PAH, and probably for other enzymes, this domain appears to have been incorporated as a handy, flexible small module with the potential to provide allosteric regulation via transmission of finely tuned conformational changes, not necessarily initiated by regulatory ligand binding at the domain itself.

Glucocorticoid-Induced Hypertension and Tetrahydrobiopterin (BH4), a Common Cofactor for the Production of Vasoactive Molecules

Excess glucocorticoids, whether produced endogenously or over-prescribed for immunosupression and antiinflammation, can lead to hypertension and cardiovascular disease. Humans and animals with glucocorticoid-induced hypertension exhibit reduced nitric oxide (NO) and serotonin, and have increased sensitivity to catecholamines. The common cofactor for the production of these vasoactive molecules is tetrahydrobiopterin (BH4). Recent research has focused on the effects of excess glucocorticoids on BH4 biosynthesis because reduced BH4 cofactor levels can alter the production of NO, serotonin, and catecholamines by NO synthase and the aromatic amino acid hydroxylases. This review will focus on the mechanisms and consequences of excess glucocorticoids on the BH4 biosynthesis pathway and the enzymes that utilize BH4 as a cofactor. Alterations in the production of BH4 contribute to glucocorticoid-induced hypertension and an understanding of the mechanisms may provide therapeutic targets to either develop synthetic glucocorticoids that do not affect BH4 biosynthesis or increase BH4 levels in conditions where glucocorticoids are elevated. Keywords: gtp cyclohydrolase 1, tetrahydrobiopterin, glucocorticoids, endothelial nitric oxide synthase, endothelium

Tetrahydrobiopterin Binding to Aromatic Amino Acid Hydroxylases. Ligand Recognition and Specificity

The three aromatic amino acid hydroxylases (phenylalanine, tyrosine, and tryptophan hydroxylase) and nitric oxide synthase (NOS) all utilize (6R)-l-erythro-5,6,7,8-tetrahydrobiopterin (BH(4)) as cofactor. The pterin binding site in the three hydroxylases is well conserved and different from the binding site in NOS. The structures of phenylalanine hydroxylase (PAH) and of NOS in complex with BH(4) are still the only crystal structures available for the reduced cofactor-enzyme complexes. We have studied the enzyme-bound and free conformations of BH(4) by NMR spectroscopy and molecular docking into the active site of the three hydroxylases, using endothelial NOS as a comparative probe. We have found that the dihydroxypropyl side chain of BH(4) adopts different conformations depending on which hydroxylase it interacts with. All the bound conformations are different from that of BH(4) free in solution at neutral pH. The different bound conformations appear to result from specific interactions with nonconserved amino acids at the BH(4) binding sites of the hydroxylases, notably the stretch 248-251 (numeration in PAH) and the residue corresponding to Ala322 in PAH, i.e., Ser in TH and Ala in TPH. On the basis of analysis of molecular interaction fields, we discuss the selectivity determinants for each hydroxylase and explain the high-affinity inhibitory effect of 7-tetrahydrobiopterin specifically for PAH.

Phenylalanine 4-Monooxygenase and the S-Oxidation of S-Carboxymethyl-L-cysteine

The identity of the enzyme responsible for the S-oxidation of the mucolytic S-substituted L-cysteine drug, S-carboxymethyl-L-cysteine (SCMC), has been actively investigated for the last 10 years. A genetic polymorphism exists in the oxidation of the thioether moiety that has been identified as a disease susceptibility factor in a number of degenerative diseases. This polymorphism has also been implicated in the wide variation in clinical response to SCMC therapy in man. To date little is known about the molecular enzymology of this reaction but a previous investigation revealed that rat activated phenylalanine 4-monooxygenase (PAH) could S-oxidise both Met- and S-methyl-L-cysteine (SMC) to their S-oxide metabolites. We have investigated the hypothesis that SCMC was also a substrate for activated PAH in the cytosolic faction of the Wistar rat. 1. Substrate and inhibitor investigation revealed that SCMC was a substrate for activated PAH activity in vitro. 2. The large aromatic amino acid hydroxylase monoclonal antibody and the Fe3+ chelator, deferoxamine, completely inhibited both Phe and SCMC oxidation to their respective metabolites. 3. Analysis of the Dixon plots revealed that both Phe and SCMC competitively inhibited each other's oxidation. 4. Correlation studies showed that the rate of production of Tyr was positively correlated to the production of both SCMC and SMC S-oxides in 20 female Wistar rat hepatic cytosolic fractions. These results strongly support the hypothesis that PAH is the enzyme responsible for SCMC S-oxidation in the rat.

Different stabilities and denaturation pathways for structurally related aromatic amino acid hydroxylases

Different stabilities and denaturation pathways for structurally related aromatic amino acid hydroxylases

Different stabilities and denaturation pathways for structurally related aromatic amino acid hydroxylases

We have compared the urea stability of the human aromatic amino acid hydroxylases (AAAHs), key enzymes involved in neurotransmitter biosynthesis and amino acid homeostasis. Tyrosine-, tryptophan- and phenylalanine hydroxylase (TH, TPH and PAH, respectively) were transiently activated at low urea concentrations and rapidly inactivated in >3 M urea. The denaturation of TH occurred through two cooperative transitions, with denaturation midpoints of 1.41 ± 0.06 and 5.13 ± 0.05 M urea, respectively. Partially denatured human TH (hTH) retained more of its secondary structure than human PAH (hPAH), and was found to exist as tetramers, whereas hPAH dissociated into dimers. Furthermore, the urea-induced aggregation of hPAH was 100-fold higher than for hTH. These results suggest that the denatured state properties of the AAAHs contribute significantly to the stability of these enzymes and their tolerance towards missense mutations.

Role of the second coordination sphere residue tyrosine 179 in substrate affinity and catalytic activity of phenylalanine hydroxylase.

Phenylalanine hydroxylase converts phenylalanine to tyrosine utilizing molecular oxygen and tetrahydropterin as a cofactor, and belongs to the aromatic amino acid hydroxylases family. The catalytic domains of these enzymes are structurally similar. According to recent crystallographic studies, residue Tyr179 in Chromobacterium violaceum phenylalanine hydroxylase is located in the active site and its hydroxyl oxygen is 5.1 A from the iron, where it has been suggested to play a role in positioning the pterin cofactor. To determine the catalytic role of this residue, the point mutants Y179F and Y179A of phenylalanine hydroxylase were prepared and characterized. Both mutants displayed comparable stability and metal binding to the native enzyme, as determined by their melting temperatures in the presence and absence of iron. The catalytic activity ( k(cat)) of the Y179F and Y179A proteins was lower than wild-type phenylalanine hydroxylase by an order of magnitude, suggesting that the hydroxyl group of Tyr179 plays a role in the rate-determining step in catalysis. The K(M) values for different tetrahydropterin cofactors and phenylalanine were decreased by a factor of 3-4 in the Y179F mutant. However, the K(M) values for different pterin cofactors were slightly higher in the Y179A mutant than those measured for the wild-type enzyme, and, more significantly, the K(M) value for phenylalanine was increased by 10-fold in the Y179A mutant. By the criterion of k(cat)/ K(Phe), the Y179F and Y179A mutants display 10% and 1%, respectively, of the activity of wild-type phenylalanine hydroxylase. These results are consistent with Tyr179 having a pronounced role in binding phenylalanine but a secondary effect in the formation of the hydroxylating species. In conjunction with recent crystallographic analyses of a ternary complex of phenylalanine hydroxylase, the reported findings establish that Tyr179 is essential in maintaining the catalytic integrity and phenylalanine binding of the enzyme via indirect interactions with the substrate, phenylalanine. A model that accounts for the role of Tyr179 in binding phenylalanine is proposed.

Perturbed 6-tetrahydrobiopterin recycling via decreased dihydropteridine reductase in vitiligo: more evidence for H2O2 stress.

To date there is ample evidence that patients with vitiligo accumulate millimolar concentrations of hydrogen peroxide (H2O2) in their epidermis as well as in their blood lymphocytes/monocytes. Several enzymes are affected by this H2O2 including catalase, glutathione peroxidase, and 4 alpha-carbinolamine dehydratase. The latter enzyme disrupts the recycling of the essential cofactor (6R)-L-erythro-5,6,7,8-tetrahydrobiopterin (6BH4) for the aromatic amino acid hydroxylases as well as the nitric oxide synthases. In this report we have elucidated the influence of H2O2 on dihydropteridine reductase (DHPR), the last enzyme in the 6BH4-recycling process. Here we show for the first time that concentrations of less than 30 microM H2O2 increase DHPR activities, whereas levels greater than 30 microM H2O2 deactivate the enzyme based on the oxidation of Met146 and Met151 in the sequence, consequently leading to disruption of the NADH-dependent enzyme active site. This oxidation was confirmed by Fourier transform-Raman spectroscopy yielding the expected SO band at 1025 cm-1 characteristic of methionine sulfoxide. Hence these results unmasked a novel regulatory mechanism for DHPR enzyme activity. Moreover, we also demonstrated that DHPR activities in whole blood of patients with vitiligo are significantly decreased in untreated patients, whereas activities are normalized after removal of epidermal H2O2 with a topical pseudocatalase (PC-KUS). Taken together, these new data add more evidence to a systemic involvement of H2O2 in the pathomechanism of vitiligo.

Low tetrahydrobiopterin biosynthetic capacity of human monocytes is caused by exon skipping in 6-pyruvoyl tetrahydropterin synthase

Biosynthesis of (6 R )-5,6,7,8-tetrahydro-L-biopterin (H(4)-biopterin), an essential cofactor for aromatic amino acid hydroxylases and NO synthases, is effectively induced by cytokines in most of the cell types. However, human monocytes/macrophages form only a little H(4)-biopterin, but release neopterin/7,8-dihydroneopterin instead. Whereas 6-pyruvoyl tetrahydropterin synthase (PTPS) activity, the second enzyme of H(4)-biopterin biosynthesis, is hardly detectable in these cells, PTPS mRNA levels were comparable with those of cell types containing intact PTPS activity. By screening a THP-1 cDNA library, we identified clones encoding the entire open reading frame (642 bp) as well as clones lacking the 23 bp exon 3, which results in a premature stop codon. Quantification of the two mRNA species in different cell types (blood-derived cells, fibroblasts and endothelial cells) and cell lines showed that the amount of exon-3-containing mRNA is correlated closely to PTPS activity. The ratio of exon-3-containing to exon-3-lacking PTPS mRNA is not affected by differential mRNA stability or nonsense-mediated mRNA decay. THP-1 cells transduced with wild-type PTPS cDNA produced H(4)-biopterin levels and expressed PTPS activities and protein amounts comparable with those of fibroblasts. We therefore conclude that exon 3 skipping in transcription rather than post-transcriptional mechanisms is a major cause of the low PTPS protein expression observed in human macrophages and related cell types.

Tetrahydrobiopterin is synthesized from 6-pyruvoyl-tetrahydropterin by the human aldo-keto reductase AKR1 family members

Tetrahydrobiopterin (BH 4) is a cofactor for aromatic amino acid hydroxylases and nitric oxide synthase. The biosynthesis includes two reduction steps catalyzed by sepiapterin reductase. An intermediate, 6-pyruvoyltetrahydropterin (PPH 4) is reduced to 1 ′-oxo-2 ′-hydroxypropyl-tetrahydropterin (1 ′-OXPH 4) or 1 ′-hydroxy-2 ′-oxopropyl-tetrahydropterin (2 ′-OXPH 4), which is further converted to BH 4. However, patients with sepiapterin reductase deficiency show normal urinary excretion of pterins without hyperphenylalaninemia, suggesting that other enzymes catalyze the two reduction steps. In this study, the reductase activities for the tetrahydropterin intermediates were examined using several human recombinant enzymes belonging to the aldo-keto reductase (AKR) family and short-chain dehydrogenase/reductase (SDR) family. In the reduction of PPH 4 by AKR family enzymes, 2 ′-OXPH 4 was formed by 3α-hydroxysteroid dehydrogenase type 2, whereas 1 ′-OXPH 4 was produced by aldose reductase, aldehyde reductase, and 20α-hydroxysteroid dehydrogenase, and both 1 ′-OXPH 4 and 2 ′-OXPH 4 were detected as the major and minor products by 3α-hydroxysteroid dehydrogenases (types 1 and 3). The activities of aldose reductase and 3α-hydroxysteroid dehydrogenase type 2 (106 and 35 nmol/mg/min, respectively) were higher than those of the other enzymes (0.2–4.0 nmol/mg/min). Among the SDR family enzymes, monomeric carbonyl reductase exhibited low 1 ′-OXPH 4-forming activity of 5.0 nmol/mg/min, but l-xylulose reductase and peroxisomal tetrameric carbonyl reductase did not form any reduced product from PPH 4. Aldose reductase reduced 2 ′-OXPH 4 to BH 4, but the other enzymes were inactive towards both 2 ′-OXPH 4 and 1 ′-OXPH 4. These results indicate that the tetrahydropterin intermediates are natural substrates of the human AKR family enzymes and suggest a novel alternative pathway from PPH 4 to BH 4, in which 3α-hydroxysteroid dehydrogenase type 2 and aldose reductase work in concert.