BH4 (6R-L-erythro-5,6,7,8-tetrahydrobiopterin) is an essential cofactor of a set of enzymes that are of central metabolic importance, including four aromatic amino acid hydroxylases, alkylglycerol mono-oxygenase and three NOS (NO synthase) isoenzymes. Consequently, BH4 is present in probably every cell or tissue of higher organisms and plays a key role in a number of biological processes and pathological states associated with monoamine neurotransmitter formation, cardiovascular and endothelial dysfunction, the immune response and pain sensitivity. BH4 is formed de novo from GTP via a sequence of three enzymatic steps carried out by GTP cyclohydrolase I, 6-pyruvoyltetrahydropterin synthase and sepiapterin reductase. An alternative or salvage pathway involves dihydrofolate reductase and may play an essential role in peripheral tissues. Cofactor regeneration requires pterin-4a-carbinolamine dehydratase and dihydropteridine reductase, except for NOSs, in which the BH4 cofactor undergoes a one-electron redox cycle without the need for additional regeneration enzymes. With regard to the regulation of cofactor biosynthesis, the major controlling point is GTP cyclohydrolase I. BH4 biosynthesis is controlled in mammals by hormones and cytokines. BH4 deficiency due to autosomal recessive mutations in all enzymes, except for sepiapterin reductase, has been described as a cause of hyperphenylalaninaemia. A major contributor to vascular dysfunction associated with hypertension, ischaemic reperfusion injury, diabetes and others, appears to be an effect of oxidized BH4, which leads to an increased formation of oxygen-derived radicals instead of NO by decoupled NOS. Furthermore, several neurological diseases have been suggested to be a consequence of restricted cofactor availability, and oral cofactor replacement therapy to stabilize mutant phenylalanine hydroxylase in the BH4-responsive type of hyperphenylalaninaemia has an advantageous effect on pathological phenylalanine levels in patients.

Tetrahydrobiopterin: biochemistry and pathophysiology.

Carbonyl reduction of aldehydes, ketones, and quinones to their corresponding hydroxy derivatives plays an important role in the phase I metabolism of many endogenous (biogenic aldehydes, steroids, prostaglandins, reactive lipid peroxidation products) and xenobiotic (pharmacologic drugs, carcinogens, toxicants) compounds. Carbonyl-reducing enzymes are grouped into two large protein superfamilies: the aldo-keto reductases (AKR) and the short-chain dehydrogenases/reductases (SDR). Whereas aldehyde reductase and aldose reductase are AKRs, several forms of carbonyl reductase belong to the SDRs. In addition, there exist a variety of pluripotent hydroxysteroid dehydrogenases (HSDs) of both superfamilies that specifically catalyze the oxidoreduction at different positions of the steroid nucleus and also catalyze, rather nonspecifically, the reductive metabolism of a great number of nonsteroidal carbonyl compounds. The present review summarizes recent findings on carbonyl reductases and pluripotent HSDs of the SDR protein superfamily.

Carbonyl reductases and pluripotent hydroxysteroid dehydrogenases of the short-chain dehydrogenase/reductase superfamily.

Classic tetrahydrobiopterin (BH4) deficiencies are characterized by hyperphenylalaninemia and deficiency of monoamine neurotransmitters. In this article, we report two patients with progressive psychomotor retardation, dystonia, severe dopamine and serotonin deficiencies (low levels of 5-hydroxyindoleacetic and homovanillic acids), and abnormal pterin pattern (high levels of biopterin and dihydrobiopterin) in cerebrospinal fluid. Furthermore, they presented with normal urinary pterins and without hyperphenylalaninemia. Investigation of skin fibroblasts revealed inactive sepiapterin reductase (SR), the enzyme catalyzing the final two-step reaction in the biosynthesis of BH4. Mutations in the SPR gene were detected in both patients and their family members. One patient was homozygous for a TC→CT dinucleotide exchange, predicting a truncated SR (Q119X). The other patient was a compound heterozygote for a genomic 5-bp deletion (1397–1401delAGAAC) resulting in abolished SPR-gene expression and an A→G transition leading to an R150G amino acid substitution and to inactive SR as confirmed by recombinant expression. The absence of hyperphenylalaninemia and the presence of normal urinary pterin metabolites and of normal SR-like activity in red blood cells may be explained by alternative pathways for the final two-step reaction of BH4 biosynthesis in peripheral and neuronal tissues. We propose that, for the biosynthesis of BH4 in peripheral tissues, SR activity may be substituted by aldose reductase (AR), carbonyl reductase (CR), and dihydrofolate reductase, whereas, in the brain, only AR and CR are fully present. Thus, autosomal recessive SR deficiency leads to BH4 and to neurotransmitter deficiencies without hyperphenylalaninemia and may not be detected by neonatal screening for phenylketonuria.

Mutations in the Sepiapterin Reductase Gene Cause a Novel Tetrahydrobiopterin-Dependent Monoamine-Neurotransmitter Deficiency without Hyperphenylalaninemia

Tetrahydrobiopterin (BH(4)) deficiencies are a highly heterogeneous group of disorders with several hundred patients, and so far a total of 193 different mutant alleles or molecular lesions identified in the GTP cyclohydrolase I (GTPCH), 6-pyruvoyl-tetrahydropterin synthase (PTPS), sepiapterin reductase (SR), carbinolamine-4a-dehydratase (PCD), or dihydropteridine reductase (DHPR) genes. The spectrum of mutations causing a reduction in one of the three biosynthetic (GTPCH, PTPS, and SR) or the two regenerating enzymes (PCD and DHPR) is tabulated and reviewed. Furthermore, current genomic variations or SNPs are also compiled. Mutations in GCH1 are scattered over the entire gene, and only 5 out of 104 mutant alleles, present in a homozygous state, are reported to cause the autosomal recessive form of inheritable hyperphenylalaninemia (HPA) associated with monoamine neurotransmitter deficiency. Almost all other 99 different mutant alleles in GCH1 are observed together with a wild-type allele and cause Dopa-responsive dystonia (DRD, Segawa disease) in a dominant fashion with reduced penetrance. Compound heterozygous or homozygous mutations are spread over the entire genes for PTS with 44 mutant alleles, for PCBD with nine mutant alleles, and for QDPR with 29 mutant alleles. These mutations cause an autosomal recessive inherited form of HPA, mostly accompanied by a deficiency of the neurotransmitters dopamine and serotonin. Lack of sepiapterin reductase activity, an autosomal recessive variant of BH(4) deficiency presenting without HPA, was diagnosed in patients with seven different mutant alleles in the SPR gene in exons 2 or 3 or in intron 2. Details on all mutations presented here are constantly updated in the BIOMDB database (www.bh4.org).

Mutations in the BH4-metabolizing genes GTP cyclohydrolase I, 6-pyruvoyl-tetrahydropterin synthase, sepiapterin reductase, carbinolamine-4a-dehydratase, and dihydropteridine reductase

Genome-wide identification of cuticular protein genes in the silkworm, Bombyx mori.

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

Tetrahydrobiopterin (BH 4 ) acts as a cofactor of the aromatic amino-acid hydroxylases, and its deficiency may result in hyperphenylalaninemia (HPA) and decreased production of the neurotransmitters. BH 4 is synthesized by sepiapterin reductase (SPR) from 6-pyruvoyl-tetrahydropterin (PPH 4 ). A patient with SPR deficiency shows no HPA; however, an SPR knockout mouse exhibits HPA. We have reported on the SPR-unrelated novel biosynthetic pathway from PPH 4 to BH 4 (salvage pathway II) in which 3α-hydroxysteroid dehydrogenase type 2 and aldose reductase work in concert. In this study, we performed the expression analysis of both proteins in humans and wild-type mice. The results of expression analysis indicated that salvage pathway II worked in human liver; however, it did not act in human brain or in mouse liver and brain. For this reason, a patient with SPR deficiency may show progressive neurological deterioration without HPA, and SPR knockout mice may exhibit HPA and abnormal locomotion activity.

Expression Analysis of the Aldo-Keto Reductases Involved in the Novel Biosynthetic Pathway of Tetrahydrobiopterin in Human and Mouse Tissues

We previously reported the purification of an ommin-binding protein (OMBP) from an acid-methanol extract of diapause eggs of the silkworm and that OMBP reacted with the anti-30K proteins antiserum. In order to clarify the relationship between OMBP and the 30K proteins, we attempted to determine the sequence of the N-terminal amino acid of OMBP, which was separated by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE). We observed ten protein spots of various isoelectric points; the spots corresponded with 30 kDa. Based on the sequence of the N-terminal amino acid (20 residues), the spots belonged to two kinds of 30K proteins (6G1 and 19G1), which are known as the major plasma proteins in the larval hemolymph of the silkworm. The proteins are expected to attach to polysaccharide because they reacted with concanavalin A and elderberry bark lectin. Immunohistochemical observations clarified that the proteins were localized in yolk granules and serosa in the diapause egg. These results suggest that OMBP is composed of 30K proteins which were modified with polysaccharides. In addition, the expression of 30K proteins mRNA was observed at early embryonic stage in diapause eggs by RT-PCR analysis. The 30K proteins as OMBP may play an important role in the transport and accumulation of tryptophan metabolites and ommochrome during the formation of serosa.

Molecular properties and tissue distribution of 30K proteins as ommin-binding proteins from diapause eggs of the silkworm, Bombyx mori

A novel RNA helicase-like protein during early embryonic development in silkworm Bombyx mori: molecular characterization and intracellular localization.

Teruhiko Iino

Papers

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

Expression Analysis of the Aldo-Keto Reductases Involved in the Novel Biosynthetic Pathway of Tetrahydrobiopterin in Human and Mouse Tissues

Molecular properties and tissue distribution of 30K proteins as ommin-binding proteins from diapause eggs of the silkworm, Bombyx mori

A novel RNA helicase-like protein during early embryonic development in silkworm Bombyx mori: molecular characterization and intracellular localization.

Properties of ommochrome-binding proteins from the pigment granules in epidermal cells of the silkworm, Bombyx mori