Current knowledge distinguishes three classes of tetrahydrobiopterin-dependent enzymes as based on protein sequence similarity. These three protein sequence clusters hydroxylate three types of substrate atoms and use three different forms of iron for catalysis. The first class to be discovered was the aromatic amino acid hydroxylases, which, in mammals, include phenylalanine hydroxylase, tyrosine hydroxylase, and two isoforms of tryptophan hydroxylases. The protein sequences of these tetrahydrobiopterin-dependent aromatic amino acid hydroxylases are significantly similar, and all mammalian aromatic amino acid hydroxylases require a non-heme-bound iron atom in the active site of the enzyme for catalysis. The second classes of tetrahydrobiopterin-dependent enzymes to be characterized were the nitric oxide synthases, which in mammals occur as three isoforms. Nitric oxide synthase protein sequences form a separate cluster of homologous sequences with no similarity to aromatic amino acid hydroxylase protein sequences. In contrast to aromatic amino acid hydroxylases, nitric oxide synthases require a heme-bound iron for catalysis. The alkylglycerol monooxygenase protein sequence was the most recent to be characterized. This sequence shares no similarity with aromatic amino acid hydroxylases and nitric oxide synthases. Motifs contained in the alkylglycerol monooxygenase protein sequence suggest that this enzyme may use a di-iron center for catalysis.
The present article is based on the Gowland Hopkins Award lecture given by the author on the 15th International Symposium on Pterins and Folates, May 9–13, 2012, in Antalya, Turkey. For a more detailed review on biochemistry and pathophysiology of tetrahydrobiopterin, the reader is referred to Ref. . Here, a short historical overview of the discovery of tetrahydrobiopterin-dependent enzymes is presented, followed by a summary of the biochemical properties of these enzymes. The reactions catalyzed by the three classes of tetrahydrobiopterin-dependent enzymes are detailed in Figure 1.
Phenylalanine hydroxylase was the first enzyme characterized to be dependent on a tetrahydropterin . It then took five more years to identify the nature of the endogenous cofactor as tetrahydrobiopterin . Soon after, the two other tetrahydrobiopterin-dependent aromatic amino acid hydroxylases, i.e., tyrosine hydroxylase (EC 184.108.40.206)  and tryptophan hydroxylase (EC 220.127.116.11) , were characterized to depend on tetrahydrobiopterin. These enzymes are required for the degradation of phenylalanine as well as for the biosynthesis of catecholamine and serotonin neurotransmitters. Studying the formation of serotonin in tryptophan hydroxylase (TPH1) knockout mice led to the discovery of the second isoform of tryptophan hydroxylase (TPH2) in 2003 . The analysis of the protein sequences of the enzymes showed significant homologies between these tetrahydrobiopterin-dependent aromatic amino acid hydroxylases, and structural and mechanistic studies revealed a general similarity of these enzymes with only subtle differences [7, 8].
In 1964, a tetrahydropteridine-dependent enzyme system for the cleavage of glyceryl ethers was first described . The responsible enzyme, alkylglycerol monooxygenase (EC 18.104.22.168.), is a very labile integral membrane protein that could not be purified up to now. Sequence assignment was achieved in 2010 by bioinformatic selection of candidate genes and recombinant expression in Chinese hamster ovary cells . This sequence turned out to be unrelated to the other groups of tetrahydrobiopterin-dependent enzymes, the aromatic amino acid hydroxylases and the nitric oxide synthases (EC 22.214.171.124). Alkylglycerol monooxygenase is the only enzyme known to cleave the ether bond of alkylglycerols, which are major membrane constituents, e.g., in the brain. The platelet-activating factor, a very potent inflammatory mediator, is also an alkylglycerol derivative. The precise physiological role of alkylglycerol monooxygenase remains to be elucidated. Current knowledge on alkylglycerol monooxygenase is summarized in Ref. .
In the late 1980s, a new class of tetrahydrobiopterin-dependent enzymes, the nitric oxide synthases, was discovered [12–14], which, from a biochemical point of view, behaves quite differently from the currently known tetrahydrobiopterin-dependent enzymes (see below). Nitric oxide synthases occur in mammals in three isoforms encoded by three different genes. They serve a variety of important functions, including neurotransmission, vasorelaxation, and host defense to pathogens and tumors [15–17]. Their protein sequences share significant sequence homology but are unrelated to the two other sequence clusters of tetrahydrobiopterin-dependent enzymes, the aromatic amino acid hydroxylases and the alkylglycerol monooxygenase. The role of tetrahydrobiopterin in the three isoforms is very similar, with only subtle differences [18, 19].
A summary of the current knowledge on the biochemistry of the three classes of tetrahydrobiopterin-dependent enzymes is presented in Table 1; for a more detailed description, the reader is referred to Ref. . The entries for the alkylglycerol monooxygenase are based on current knowledge, but they should be treated with caution because no pure enzyme has been prepared so far to study the biochemical parameters. These were observed mainly in rat liver microsomal preparations or in homogenates of transiently transfected Chinese hamster ovary cells.
|Aromatic amino acid hydroxylases||Alkylglycerol monooxygenase||Nitric oxide synthases (NOS)|
|Members in humans (gene symbols)||Phenylalanine hydroxylase (PAH)||Alkylglycerol monooxygenase (AGMO)||Neuronal NOS (NOS1)|
|Tyrosine hydroxylase (TH)||Inducible NOS (NOS2)|
|Tryptophan hydroxylases (TPH1, TPH2)||Endothelial NOS (NOS3)|
|Substrate atom hydroxylated (cf. Figure 1)||Aromatic carbon atom||Aliphatic carbon atom||Guanidino nitrogen atom|
|Affinity of tetrahydrobiopterin (Km)||~30 µM||~3 µM||~0.3 µM|
|Recycling of tetrahydrobiopterin||External (PCDB1+QDPR)||External [PCBD1 (?)+QDPR]||Internal|
|Chemistry of cofactor assisted catalysis||Two-electron||Two-electron||One-electron|
|Selectivity for the side chain in position 6 of the pterin||Medium||Medium||High|
|Form of the catalytically active iron||Non-heme||Di-iron center||Heme|
|Oxygen activation by tetrahydrobiopterin||Yes||Yes||No|
|Soluble – membrane association||Soluble||Integral membrane protein||Soluble (NOS3 myristoylated and palmitoylated)|
|Distinct sequence cluster||Aromatic amino acid hydroxylases||Alkylglycerol monooxygenases||Nitric oxide synthases|
|Structure||Crystal structures available||Only a predicted structure of a part of the protein available||Crystal structures of oxygenase domains available|
Alkylglycerol monooxygenase could not be purified so far, and the enzymatic characteristics are therefore based on measurements in rat liver microsomes or in homogenates of transfected cells. The two recycling enzymes of tetrahydrobiopterin are shown by their gene symbols in the table.
The three classes of tetrahydrobiopterin-dependent enzymes all catalyze a mixed-function oxygenase reaction, i.e., one oxygen atom of the oxygen molecule is incorporated into the substrate, whereas the other one is converted to water (Figure 1). However, the nature of the substrate atom hydroxylated is different for each class of tetrahydrobiopterin-dependent enzymes. It is an aromatic carbon atom in the aromatic amino acid hydroxylases, a guanidino nitrogen in nitric oxide synthases, and an aliphatic carbon atom in alkylglycerol monooxygenase. The affinity for the tetrahydrobiopterin cofactor varies over two orders of magnitude, being highest in the nitric oxide synthases and lowest in the aromatic amino acid hydroxylases. In addition to the highest affinity for tetrahydrobiopterin, nitric oxide synthases are also the class of enzymes with the highest selectivity for the 6R-1′,2′-dihydroxypropyl side chain of tetrahydrobiopterin. Although nitric oxide synthases are almost exclusively stimulated by tetrahydrobiopterin, with the action of other tetrahydropterins only at orders-of-magnitude-higher concentrations, aromatic amino acid hydroxylases and alkylglycerol monooxygenase can be stimulated with some other tetrahydropterins to a more comparable extent. Aromatic amino acid hydroxylases and presumably also alkylglycerol monooxygenase use tetrahydrobiopterin for oxygen activation. This is not the case for nitric oxide synthases . The tetrahydrobiopterin cofactor leaves the enzymatic reaction of aromatic amino acid hydroxylases as 4a-hydroxy derivative in aromatic amino acid hydroxylases (and presumably also in alkylglycerol monooxygenase). This compound is recycled by pterin 4a-carbinolamine dehydratase (PCBD1) and quinoid dihydropteridine reductase (QDPR) back to the active tetrahydrobiopterin cofactor (reviewed in Ref. ). In contrast, in nitric oxide synthases, the cofactor is thought to undergo only one-electron transformations to a tetrahydrobiopterin radical, which is regenerated at the enzyme without the aid of additional external enzymes. All three classes of tetrahydrobiopterin-dependent enzymes contain an iron, which is essential for catalysis, but in a different form. This is a non-heme iron bound to two histidines and a glutamate in aromatic amino acid hydroxylases and a heme-bound iron in nitric oxide synthases. The occurrence of the fatty acid hydroxylase motif in the protein sequence of alkylglycerol monooxygenase suggests that this enzyme may contain a di-iron center in its active site . This feature may be linked to the exceptional instability of this enzyme because no protein containing the fatty acid hydroxylase motif has ever been purified in its active form so far . Another feature related to experimental difficulties with handling alkylglycerol monooxygenase is that it is an integral membrane protein occurring in the endoplasmic reticulum , which has nine transmembrane regions , whereas the other tetrahydrobiopterin-dependent enzymes are soluble proteins. One of these, endothelial nitric oxide synthase, is myristoylated and palmitoylated post-translationally, targeting it to the plasmalemmal caveolae . The crystal structures of the aromatic amino acid hydroxylases (reviewed in Refs. [7, 8]) and of the oxygenase domains of the three nitric oxide synthases (reviewed in Ref. ) are available. For alkylglycerol monooxygenase, only a predicted structure of a part of the protein has been suggested .
The three classes of tetrahydrobiopterin-dependent enzymes are encoded by proteins belonging to three different sequence clusters; they use three different forms of iron for catalysis; and they hydroxylate three different kinds of substrate atoms, all in monooxygenase-type reactions. Although the biochemistry of tetrahydrobiopterin in alkylglycerol monooxygenase and in aromatic amino hydroxylases shares many features, nitric oxide synthases use tetrahydrobiopterin in a considerably different manner.
Funding: Our experimental work was funded by the Austrian Science Funds (fwf), project P22406.
1. Werner ER, Blau N, Thony B. Tetrahydrobiopterin: biochemistry and pathophysiology. Biochem J 2011;438:397–414. Search in Google Scholar
2. Kaufman S. The participation of tetrahydrofolic acid in the enzymic conversion of phenylalanine to tyrosine. Biochim Biophys Acta 1958;27:428–9. Search in Google Scholar
3. Kaufman S. The structure of the phenylalanine-hydroxylation cofactor. Proc Natl Acad Sci USA 1963;50:1085–93. Search in Google Scholar
4. Nagatsu T, Levitt M, Udenfriend S. Tyrosine hydroxylase. The initial step in norepinephrine biosynthesis. J Biol Chem 1964;239:2910–7. Search in Google Scholar
5. Lovenberg W, Jequier E, Sjoerdsma A. Tryptophan hydroxylation: measurement in pineal gland, brainstem, and carcinoid tumor. Science 1967;155:217–9. Search in Google Scholar
6. Walther DJ, Peter JU, Bashammakh S, Hörtnagl H, Voits M, Fink H, et al. Synthesis of serotonin by a second tryptophan hydroxylase isoform. Science 2003;299:76. Search in Google Scholar
7. Fitzpatrick PF. Mechanism of aromatic amino acid hydroxylation. Biochemistry 2003;42:14083–91. Search in Google Scholar
8. Teigen K, McKinney JA, Haavik J, Martinez A. Selectivity and affinity determinants for ligand binding to the aromatic amino acid hydroxylases. Curr Med Chem 2007;14:455–67. Search in Google Scholar
9. Tietz A, Lindberg M, Kennedy EP. A new pteridine-requiring enzyme system for the oxidation of glyceryl ethers. J Biol Chem 1964;239:4081–90. Search in Google Scholar
10. Watschinger K, Keller MA, Golderer G, Hermann M, Maglione M, Sarg B, et al. Identification of the gene encoding alkylglycerol monooxygenase defines a third class of tetrahydrobiopterin-dependent enzymes. Proc Natl Acad Sci USA 2010;107:13672–7. Search in Google Scholar
11. Watschinger K, Werner ER. Alkylglycerol monooxygenase. IUBMB Life 2013;65:366–72. Search in Google Scholar
12. Tayeh MA, Marletta MA. Macrophage oxidation of L-arginine to nitric oxide, nitrite, and nitrate. Tetrahydrobiopterin is required as a cofactor. J Biol Chem 1989;264:19654–8. Search in Google Scholar
13. Kwon NS, Nathan CF, Stuehr DJ. Reduced biopterin as a cofactor in the generation of nitrogen oxides by murine macrophages. J Biol Chem 1989;264:20496–501. Search in Google Scholar
14. Mayer B, John M, Bohme E. Purification of a Ca2+/calmodulin-dependent nitric oxide synthase from porcine cerebellum. Cofactor-role of tetrahydrobiopterin. FEBS Lett 1990;277:215–9. Search in Google Scholar
15. Forstermann U, Sessa WC. Nitric oxide synthases: regulation and function. Eur Heart J 2012;33:829–37. Search in Google Scholar
16. Alderton WK, Cooper CE, Knowles RG. Nitric oxide synthases: structure, function and inhibition. Biochem J 2001;357:593–615. Search in Google Scholar
17. Stuehr DJ. Mammalian nitric oxide synthases. Biochim Biophys Acta 1999;1411:217–30. Search in Google Scholar
18. Gorren AC, Mayer B. Nitric-oxide synthase: a cytochrome P450 family foster child. Biochim Biophys Acta 2007;1770:432–45. Search in Google Scholar
19. Tejero J, Stuehr D. Tetrahydrobiopterin in nitric oxide synthase. IUBMB Life 2013;65:358–65. Search in Google Scholar
20. Riethmuller C, Gorren AC, Pitters E, Hemmens B, Habisch HJ, Heales SJ, et al. Activation of neuronal nitric-oxide synthase by the 5-methyl analog of tetrahydrobiopterin. Functional evidence against reductive oxygen activation by the pterin cofactor. J Biol Chem 1999;274:16047–51. Search in Google Scholar
21. Shanklin J, Guy JE, Mishra G, Lindqvist Y. Desaturases: emerging models for understanding functional diversification of diiron-containing enzymes. J Biol Chem 2009;284: 18559–63. Search in Google Scholar
22. Watschinger K, Fuchs JE, Yarov-Yarovoy V, Keller MA, Golderer G, Hermetter A, et al. Catalytic residues and a predicted structure of tetrahydrobiopterin-dependent alkylglycerol mono-oxygenase. Biochem J 2012;443:279–86. Search in Google Scholar
23. Gonzalez E, Kou R, Lin AJ, Golan DE, Michel T. Subcellular targeting and agonist-induced site-specific phosphorylation of endothelial nitric-oxide synthase. J Biol Chem 2002;277: 39554–60. Search in Google Scholar
24. Daff S. NO synthase: structures and mechanisms. Nitric Oxide 2010;23:1–11. Search in Google Scholar
©2013 by Walter de Gruyter Berlin Boston
This article is distributed under the terms of the Creative Commons Attribution Non-Commercial License, which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.