Three classes of tetrahydrobiopterin-dependent enzymes

Introduction

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. [1]. 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.

Figure 1

Chemical reactions catalyzed by the three classes of tetrahydrobiopterin-dependent enzymes.

The respective gene symbols in humans are in parentheses. The intermediate semiacetal as initial reaction product of the alkylglycerol monooxygenase is assumed to occur by plausibility, an experimental proof of it as well as knowledge on whether or not its rearrangement is assisted by an additional enzyme is currently missing. The figure was modified from Ref. [1].

Discovery of tetrahydrobiopterin-dependent enzymes

Biochemical characteristics of the three classes of tetrahydrobiopterin-dependent enzymes

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. [1]. 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.

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 [20]. 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. [1]). 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 [10]. 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 [21]. Another feature related to experimental difficulties with handling alkylglycerol monooxygenase is that it is an integral membrane protein occurring in the endoplasmic reticulum [10], which has nine transmembrane regions [22], 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 [23]. 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. [24]) are available. For alkylglycerol monooxygenase, only a predicted structure of a part of the protein has been suggested [22].

Conclusion

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.