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Bh4 Synthesis Essay

Abstract

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.

Abbreviations: AGMO, alkylglycerol mono-oxygenase; ApoE, apolipoprotein E; AR, aldose reductase; BH4, 6R-L-erythro-5,6,7,8-tetrahydrobiopterin; CR, carbonyl reductase; DCoH1, dimerization cofactor of hepatocyte nuclear factor 1α; DHFR, dihydrofolate reductase; DHPR, dihydropteridine reductase; DRD, dopa-responsive dystonia; GFRP, GTP cyclohydrolase I feedback regulatory protein; GTPCH, GTP cyclohydrolase I; HNF-1α, hepatocyte nuclear factor 1α; IGF-1, insulin-like growth factor-1; ki, knockin; ko, knockout; NOS, NO synthase; PAH, phenylalanine hydroxylase; PCD, pterin-4a-carbinolamine dehydratase; PTPS, 6-pyruvoyltetrahydropterin synthase; SR, sepiapterin reductase; TH, tyrosine hydroxylase; TPH, tryptophan hydroxylase

  • © The Authors Journal compilation © 2011 Biochemical Society

We aimed at elucidating the physiological and pathological functions of CAs by using genetically engineered mice and in patients with CA dysfunction, based on the structures of the genes and deduced proteins of CA-synthesizing enzymes. Therefore, we cloned the genes of humans and mice for the enzymes related to the biosynthesis of CAs and the BH4 cofactor of TH. A summary of the structures of these genes and CA- and BH4-synthesizing enzymes in humans are shown in Table I. (see reviews 3), 22)) Multiple mRNAs are produced in humans by alternative mRNA processing from a single gene in the case of TH, AADC, DBH, and PNMT. However, except for the human TH gene, which produces 4 isoform proteins, the other enzymes are composed of a single protein.

3.1. Tyrosine 3-monooxygenase (TH): the human gene, human multiple isoforms, and regulation

In early 1964, among the four enzymes involved in CA biosynthesis, only the enzyme responsible for converting tyrosine to DOPA still remained elusive. Then in that year we discovered a pteridine-dependent monooxygenase as this elusive enzyme.15), 23) Until the discovery of TH in 1964, there were three hypotheses for the conversion of tyrosine to DOPA in CA-producing cells: a non-enzymatic reaction; monophenol monooxygenase (tyrosinase) as the possible enzyme; or the presence of an unknown enzyme. Tyrosine can be easily converted to DOPA non-enzymatically in vitro, but not in vivo; and tyrosinase produces DOPA via dopaquinone and leucodopachrome from tyrosine in melanin-producing melanocytes,24) but not in CA-producing cells. Assuming that an unknown enzyme to convert tyrosine to DOPA may exist in CA-containing tissues, at the NIH we started to work to discover such enzyme. We first developed a highly sensitive isotopic assay to detect the assumed enzyme activity; L-[14C] tyrosine with high specific radioactivity was used as a substrate, and L-[14C] DOPA, if enzymatically formed, was isolated on an alumina column and assayed by the use of a liquid scintillation counter. We started our initial work to discover the enzyme in tissue slices and minces of the rat brain stem, where the dopamine content is high and the tissue should contain the enzyme and all of the necessary cofactors. We found the absolute stereo-specificity of this enzyme, which permitted the use of D-[14C] tyrosine as a control, and we became convinced that we were really detecting a new enzyme. We found that the bovine adrenal medulla contained a large amount of the enzyme in a soluble fraction, and developed a new and rapid assay for the activity by using L-[3,4-3H] tyrosine as substrate to determine the amount of 3H released in the hydroxylation reaction at the 3-position.25) Thus we could partially purify the enzyme. After testing many probable cofactor substances, the preparations were shown to require a tetrahydropteridine and molecular oxygen for the enzyme activity. Thus the systematic name of the enzyme is tyrosine 3-monooxygenase. Ferrous iron was also found to be another essential cofactor.15) This enzyme was later found by Levitt et al. to be rate-limiting in vivo in the biosynthesis of CAs.

TH has been shown to be regulated by complex mechanisms (see reviews 26)–28)). The enzyme is inhibited by dopamine and various catechol compounds, and so we proposed feedback inhibition as the mechanism for the short-term regulation of CA biosynthesis.15) Dopamine not only acutely inhibits TH activity competitively with a pteridine cofactor, but also inactivates the enzyme activity by binding to the protein.28) Among various catechol- and tyrosine-derivative inhibitors, L-α-methyl-p-tyrosine is the most potent competitive inhibitor toward the substrate L-tyrosine29); and it has been widely used to inhibit the enzyme in vivo in experimental animals. Several natural inhibitors of TH were found to be produced by microorganisms in the search for microbial enzyme inhibitors by Umezawa et al.; and oudenone was one of them.30) It inhibits the enzyme competitively with respect to the pteridine cofactor.

TH is regulated in the short term not only by feed-back inhibition but also by phosphorylation and dephosphorylation by a complex mechanism, as described below in more detail. It is phosphorylated at Ser8, Ser19, Ser31 and Ser40. Activation of TH by phosphorylation of the enzyme is mainly catalyzed by protein kinase A, Ca2+/calmodulin-dependent protein kinase II (Ca/CaMPK II), and protein kinase C.27), 28) These protein kinases activate TH by phosphorylation in the short term, and also increase production of the protein in the long term. Medium-to long-term regulation of the enzyme activity occurs at various phases of gene expression, such as transcription, alternative RNA processing, RNA stability, and translation. The enzyme activity can also be regulated at the level of protein stability, which is increased by an increase in the intracellular concentration of BH4.19)

Under stress this enzyme protein is increased by induction. The first indication of the induction of TH was found by the increased turn over of noradrenaline and the increased maximum velocity of this enzyme in the sympathetic nerves in the heart of sino-aortic denervated rabbits.31) Induction of TH was also confirmed after chemical sympathectomy as a compensatory mechanism of noradrenaline depletion.32) TH is induced under chronic stress together with DBH and PNMT.

Purification of TH was difficult, but was finally achieved in early 1980’s from various tissues, e.g., bovine adrenal medulla,33) rat adrenals,34) and human brain and adrenals.35)

The primary structure of the enzyme from various species including humans was determined by cloning of cDNA after the 1980’s (see reviews 3), 36), 37)). TH from various mammalian species is a 240-kDa homotetramer composed of four identical 60-kDa subunits. Each subunit has a C-terminal catalytic domain that binds the substrates tyrosine and molecular oxygen and the tetrahydropteridine cofactor with ferrous iron, and an N-terminal regulatory domain containing phosphorylated serine residues (Fig. 4). TH from non-primate animals such as mice38) is a single protein. Only human TH has four isoform types1–4 (hTH1, hTH2, hTH3, and hTH4), due to alternative mRNA splicing.39)–42) By comparison of the genomic DNA sequences of various primates, we found that non-human primates such as Macaca irus and Macaca fuscata (Japanese monkeys),43) gibbon, orangutan, gorilla, and chimpanzee44) produce only two of the TH isoforms, corresponding to human TH types 1 and 2 (hTH1 and hTH2). In contrast to humans, monkeys, like non-primate mammals, lack exon 2 in humans, but they have two isoforms corresponding to hTH1 and hTH2 by alternative mRNA splicing of exon 1. The expression of human TH types 1–4 and monkey TH types 1 and 2 was proved immunohistochemically by Haycock.45)

Fig. 4.

A Structures of the human tyrosine 3-monooxygenase (tyrosine hydroxylase, TH) genes, indicating the alternative splicing pathway producing the four types of human tyrosine 3-monooxygenase (hTH1–hTH4) mRNAs from a single gene. The 3′-terminal...

Crystallization of TH protein has been difficult. Goodwill et al.46) succeeded by removing the N-terminal regulatory domain: the crystal structure of the C-terminal catalytic and tetramerization domains of rat TH in the presence of the cofactor analogue 7,8-dihydrobiopterin and iron showed the mode of the pteridine cofactor binding and the proximity of its hydroxylated 4a carbon of the pteridine ring to the required iron.

The human TH gene42) is composed of 14 exons interrupted by 13 introns, spanning approximately 8.5 kb; whereas the genes of the enzyme of non-human primates and non-primate mammals lack exon 2, and consist of 13 exons. The 12-bp insertion sequence is derived from the 3′-terminal portion of exon 1 and the 81-bp insertion sequence is encoded by exon 2, which is specific in the human gene. The N-terminal region is encoded by the 5′-portion of exon 1, and the remaining region from exon 3 to exon 14 is common to all four kinds of TH mRNA. Thus, these four mRNAs differ by the presence of 12 (hTH2), 81 (hTH3), or 93 (12 plus 81, hTH4) additional nucleotides between nucleotide 90 and 91 of hTH1 (Fig. 4).3), 39)–42) Dumas et al.47) further reported three more isoforms of human TH produced by skipping of exon 3; and they found higher levels of these isoforms in the adrenal medulla of patients with progressive supranuclear palsy (PSP). We also looked for these new isoforms in the brain, but could not detect them in the brain of controls or patients with PSP. Instead we found a new splicing variant in the human adrenal medulla of a normal control; the mRNA lacked exon 4, resulting in a premature stop codon at amino acid 147.48) Although hTH1–hTH4 are the major isoforms of human TH, yet more isoforms of mRNA may exist in humans. As mentioned above (shown in the schematic diagram of Fig. 4), TH is activated by phosphorylation of mainly Ser19, Ser31, and Ser40 among the 4 Ser residues in the N-terminal regulatory domain, and deactivated by dephosphorylation via protein phosphatase (type 2A). Ser40 is mainly phosphorylated by protein kinase A, and Ser19 mainly by Ca/CaMPK II. We found that Ca/CaMPK II may phosphorylate and activate TH of PC 12h cells when they are depolarized by high K+, because a selective inhibitor of Ca/CaMPK II, KN-62 (1-[N,O-bis(5-isoquinolinsulfonyl)-N-methyl-L-tyrosyl]-4-phenylpiperazine) inhibits this TH phosphorylation and reduces dopamine synthesis.49) Itagaki et al.50) found that the chaperon 14-3-3 protein binds and activates TH at Ser19 phosphorylated by Ca/CaMPK II, indicating depolarization-evoked activation of TH in vivo; these results agree with the fact that Ca/CaMPK II mediates phosphorylation of TH by hormonal and electrical stimuli, which leads to elevation of Ca2+ levels. Lehmann et al.51) proposed human TH isoforms (hTH1–hTH4) to be differentially regulated via hierarchical phosphorylation. They reported that phosphorylation of the human enzyme type 1 (hTH1) at Ser31 by extracellular signal-regulated protein kinase (ERK; hTH2 was not phosphorylated by ERK) produced a 9-fold increase in the rate of phosphorylation of Ser40, whereas it had little effect on that rate in the TH types 3 and 4 (hTH3, hTH4). Phosphorylation of the Ser19 of hTH2 increased the phosphorylation of Ser40 more strongly than did that of the same Ser of hTH1. Thus, hTH1 might be regulated by an ERK pathway; and, hTH2, by a Ca/CaMPK II pathway. Ota with Nakashima et al.52), 53) prepared various deletion mutants of the regulatory N-terminus domain of human TH type 1 (hTH1) and found that deletion of N-terminus of the enzyme enhances the stability of the enzyme.

As described above, TH is regulated in the long term under stress by enzyme induction at the transcriptional level. Transcription of the gene is regulated by several transcription factors such as cAMP-response element binding protein (CREB), AP1, Egrl1, AP2, dyad, SP1, ATF-2, hypoxiainducible transcription factor, and Nurr1 (see review 26)). The expression of TH in cultured cells and tissues producing CAs is regulated by various first messengers: e.g., dopamine, dopamine agonists and antagonists, nicotine, vasoactive intestinal polypeptide (VIP), secretin, angiotensin II, bradykinin, neurotensin, hypoxia-inducible protein, nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), glial cell line-derived neurotrophic factor (GDNF), basic fibroblast growth factor (bFGF), ciliary neurotrophic factor (CNTF), insulin-like growth factor-I (IGF-I), and retinoic acid receptor. V-1/myotrophin containing cdc10/SWI6 motifs is unique since it induces not only TH, but also the pteridine cofactor-synthesizing enzyme, GCH1, in PC12 cells.54) Insulin-like growth factor-I (IGF-I) also induces not only TH, but also DBH and PNMT in bovine adrenomedullary chromaffin cells. These various messengers may coordinate TH induction with the induction of other CA-synthesizing enzymes under stress conditions.

3.2. Human aromatic L-amino acid decarboxylase (AADC) gene: Tissue-specific alternative splicing generates neuronal and non-neuronal types of mRNA in human AADC, but produces a single enzyme protein

AADC is the only one enzyme among the CA-synthesizing enzymes that is expressed in both neuronal CA cells and serotonin cells in the brain, and also in non-neuronal cells in the periphery such as those in the liver and kidney. Thus this enzyme relates to the biosynthesis of two important slow-acting neurotransmitters, i.e., CAs and serotonin, both of which play important roles in emotion, memory, and other higher brain functions in human behaviour. AADC requires pyridoxal phosphate as a cofactor. The enzyme is a homodimer composed of two identical 50-kDa subunits (Table I). We cloned cDNA10) of the human AADC and the genomic DNA,55) and assigned the gene to chromosome 7p12.1–p.12.3.55) The human gene, being approximately 100 kb in size and composed of 16 exons, is expressed in both CA and serotonin neurons as well as in non-neuronal tissues such as the liver. We (Ichinose et al.) found that an alternative usage of the non-neuronal (L1) and neuronal (N1) first exons in the 5′-untranslated regions of the human gene produces neuronal (CA and serotonin type) and non-neuronal (liver type) mRNAs encoding the same single protein.56), 57)

Furthermore, some neurons called D neurons in the brain express AADC only without expression of TH. The substrate of the enzyme in vivo and physiological and pathological functions of these D neurons are not clear, but they are distributed mainly near the ventricular system in the brain.58)–60) Nagatsu, I. et al.60) found that the nerve terminals of some D-neurons face the cerebral ventricle between the ependymal cells, suggesting that some monoamine neurotransmitter synthesized in the D-neurons may be released directly into cerebrospinal fluid. They also reported that, although D-neurons should synthesize some monoamine including dopamine or serotonin from various aromatic L-amino acids as the substrate, neither dopamine nor serotonin was identified in the D-neurons in the mouse and rat spinal cord by immunohitochemistry using dopamine- or serotonin-specific antibody. Thus, the physiological significance of dopamine-neurons remains to be determined by further investigation. A possible candidate of the neurotransmitter might be a trace amine in the brain such as tyramine or octopamine.

3.3. Human dopamine β-monooxygenase (DBH) gene: Alternative polyadenylation of human DBH gene produces two mRNAs, but the gene encodes a single glycoprotein with high concentrations in human blood

DBH is a copper-containing, ascorbate-requiring monooxygenase that catalyzes the hydroxylation of a phenylethylamine (mainly dopamine in vivo) at the β-C of the side chain for a phenylethanolamine (i.e., noradrenaline from dopamine), using molecular oxygen and ascorbic acid as an electron donor.14), 61) Human DBH is a 290-kDa homotetramer consisting of four subunits of Mr 64862 with 578 amino acids (603 amino acids including the signal peptide) and containing 2 atoms of Cu per subunit (Table I). DBH is a glycoprotein, as it contains carbohydrate side chains that may influence the stability of the enzyme. DBH is specifically localized in noradrenaline and adrenaline neurons (A1–A7 neurons and C1–C3 neurons) of the brain as well as in noradrenaline neurons of the peripheral sympathetic nerves and in adrenaline and noradrenaline cells of the adrenal medulla. Therefore, this monooxygenase is a marker of noradrenaline and adrenaline cells. Also DBH is the only CA-synthesizing enzyme localized in synaptic vesicles in noradrenaline and adrenaline neurons and in chromaffin granules containing adrenaline or noradrenaline in the adrenal medulla. About 50% of the activity is tightly bound to the vesicular membranes, and the rest of the activity can be easily released by hypotonic treatment of the vesicles. The soluble form of the enzyme is secreted into cerebrospinal fluid in the brain and into blood in the periphery together with noradrenaline or adrenaline as neurotransmitter or hormone.21), 62), 63) Interestingly only humans among primate and non-primate mammals have high DBH activity in their blood.62), 63); rats have very low activity.64) This may be due to a standing position of humans requiring high sympathetic nerve activity. In the blood or crude extracts of tissues, the activity is inhibited by the endogenous inhibitors that is sulfhydryl compounds like glutathione and cysteine;65) but for the assay of the activity the inhibition can be removed by N-ethylmaleimide or Cu, either of which binds with sulfhydryl groups.62), 63) Among natural inhibitors, Hidaka et al.66) discovered fusaric acid (5-butylpicolinic acid) as a specific and potent inhibitor of DBH, which have hypotensive activity in vivo.

We (Kobayashi et al.67)) isolated two different cDNAs (named types A and B based on the differences in their 3′-terminal regions) and the genomic DNA of human DBH. We showed that the two mRNAs are generated through alternative polyadenylation from a single gene. Our type A cDNA was identical to a cDNA encoding human DBH isolated by Lamouroux et al.68) The human DBH gene spans approximately 23 kb and consists of 12 exons interrupted by 11 introns. Exon 12 encodes the 3′-region of 1013 bp including the 300-bp sequence in type A. The 3′-untranslated region may be involved in mRNA stability and translational efficiency. The ratio of type A to type B mRNAs in human pheochromocytoma cells is approximately 1.0 to 0.2. We found possible transcription regulatory elements, including TATA, CCAAT, CAAAA, GC boxes, cyclic AMP response element (CRE), AP-2 element, and glucocorticoid response element (GRE), near the transcription initiation site of the human DBH gene. The cyclic AMP-mediated regulation of transcription from the DBH promoter is mediated by the AP1 proteins, i.e., c-Fos, c-Jun, and JunD. Cyclic AMP, diacylglycerol, and Ca2+ increase the transcription of both TH and DBH and lead to increased CA biosynthesis. We (Ishiguro et al.69)) identified and characterized a novel phorbol ester-response DNA sequence in the 5′-flanking region of the human DBH gene. The data suggest that transcriptional up-regulation of the human DBH gene in response to TPA (12-O-tetradecanoylphorbol-13-acetate) requires coordination among this novel TPA-response element (TRE), cyclic AMP-response element (CRE), and the YY1 binding site. We also cloned mouse DBH cDNA and the genomic DNA.70) The mouse DBH gene was composed of 12 exons about 17 kb in length, encoding a protein of 621 amino acids with Mr 70189. Typical TATA and CCAAT boxes were observed in the 5′-upstream region of the mouse gene. Northern blot analysis of adrenal gland detected a single size species of the mouse DBH mRNA.

The protein content and activity of DBH in human blood vary widely between individuals and are remarkably constant in each individual62), 63) and genetically determined. Although the activity of DBH in human plasma is specifically high among mammals, a small subgroup of the population has low activity levels. The activity in blood has been measured in various diseases. Linkage and association studies on human plasma DBH by Cubells et al.with us71) indicated the structural gene encoding DBH (locus name, DBH) to be a major quantitative trait locus for plasma DBH activity, and also to influence DBH protein levels in cerebrospinal fluid. Zabetian et al.72) further identified a new polymorphism (−1021 C→T) in the 5′-flanking region of the DBH gene as a major genetic marker for plasma DBH activity, which provides a new tool for investigation of the role of both DBH protein and the DBH gene in human diseases.

3.4. Human noradrenaline (phenylethanolamine) N-methyltransferase (PNMT) gene

PNMT is the terminal enzyme in CA biosynthesis, and catalyzes the methylation of noradrenaline to adrenaline, using S-adenosyl-L-methionine as the methyl donor.12) The enzyme is found in adrenaline cells of the adrenal medulla where adrenaline is synthesized, stored, and secreted as adrenomedullary hormone. PNMT is also localized in adrenaline (C1–C3) neurons in the medulla oblongata,1), 13) which neurons send their axons to various regions of the brain such as the hypothalamus, striatum73) and amygdala. These adrenaline neurons are supposedly involved in some important neuro-physiological functions such as cardiovascular and neuroendocrine regulation of the brain. PNMT is a 30-kDa monomeric enzyme and requires various phenylethanolamines including noradrenaline as substrates to form N-methylphenylethanolamines such as adrenaline. We (Kaneda et al.74)) cloned the full-length cDNA of the human PNMT and found it to encode a protein consisting of 282 amino acids with Mr 30853, and to reside on chromosome 17. We (Sasaoka et al.75)) also cloned the genomic DNA of the human enzyme, which consists of 3 exons and 2 introns and encodes a single protein. We observed a minor mRNA (named type B), besides the major mRNA (named type A). The type B mRNA carries an approximately 700-nucleotide-long untranslated region in its 5′ terminus, suggesting that the two types of mRNA are produced from a single gene through the use of two alternative promoters. Consensus sequences for glucocorticoid response element (GRE) and Sp1 binding sites are observed in the 5′-flanking region of the gene. We also cloned the mouse PNMT genomic DNA and cDNA for application to studies on transgenic mice. The genomic DNA spanned about 1.8 kb, and consisted of 3-exons; and the typical TATA, GC, and CACCC boxes as well as several sequences homologous to the glucocorticoid response element (GRE) were located in the 5′-flanking region.76)

3.5. Human genes for biosynthetic enzymes of tetrahydrobiopterin (BH4), the cofactor of tyrosine 3-monooxygenase (TH)

BH4 is a cofactor of TH; and it indirectly regulates the biosynthesis of CAs by regulating the activity of TH, the rate-limiting enzyme. As described above, there exist three enzymes for the biosynthesis of BH4 and two recycling enzymes during its oxidation in the TH reaction (Fig. 3, see reviews 4), 17), 22)). We isolated and characterized the human genes of GTP cyclohydrolase I (GCH1), the first and regulatory enzyme, and sepiapterin reductase (SPR), the last enzyme, of BH4 biosynthesis. Human GCH1 is encoded by three distinct mRNAs, hGCH-1, -2, and -3; and hGCH-1 is the most abundant of them in human liver.77) The full-length cDNA for mouse GCH178) encodes a protein of 241 amino acid sequence that is highly homologous to that of the human type 1 enzyme. We further characterized human and mouse GCH1 genes.79) As described below, the mutation of the human genes causes autosomal dominant GTP cyclohydrolase I deficiency/DOPA responsive dystonia (DRD)/Segawa’s disease and autosomal recessive GTP cyclohydrolase I deficiency/atypical phenylketonuria. The human GCH1 gene is composed of six exons spanning approximately 30 kb. The structural heterogeneity of human GCH1 mRNAs is caused by an alternative usage of the splicing acceptor site at the sixth exon. We also cloned cDNA80) and genomic DNA81) of the human SPR. The human cDNA encoded a protein of 261 amino acids with Mr 28047. The predicted amino acid sequence of human SPR showed a 74% identity with the sequence of the completely purified mature rat enzyme,82) the structure of which was determined by amino acid sequencing and began with an N-acetyl methionyl residue at its N-terminus. GCH1 is distributed in mice in CA neurons in the brain, adrenal medulla, and liver where BH4 is synthesized.83) SPR was proved by confocal microscopy to be colocalized with TH in the CA neurons of the human brain.84)