GTP cyclohydrolase I: purification, characterization, and effects of inhibition on nitric oxide synthase in nocardia species.

A tetrahydrobiopterin (BH4)-dependent bacterial nitric oxide synthase (NOS), designated NOSNoc, was first purified and characterized in Nocardia sp. strain NRRL 5646 in our laboratory (7). Continued pursuit of the possible roles of NOS resulted in the demonstration of guanylate cyclase (GC) activity in this bacterium (34). With viable Nocardia cells, additions of BH4 plus arginine enhanced GC activity and the amounts of cyclic GMP eightfold. This work established a novel role for NOSNoc in Nocardia similar to that known to exist in higher life forms. The occurrence of a BH4 biosynthetic pathway in Nocardia was partially established by high-performance liquid chromatography (HPLC) product analysis. Mass spectrometry and nuclear magnetic resonance spectroscopy were used to confirm the identifications of neopterin and biopterin produced by crude cell extract conversions of GTP (34). The novelty and occurrence of a defined role for BH4 in Nocardia prompted us to examine the nature of GTP cyclohydrolase I (GTPCH; EC 3.5.4.16) involved in the biosynthesis of BH4 in this organism. GTPCH catalyzes the conversion of GTP to dihydroneopterin triphosphate via a mechanistically complex ring expansion process (2, 27, 30, 32). This reaction is the first step in the biosynthetic pathway leading to the synthesis of the pteridine portion of tetrahydrofolate (FH4) in plants and some microorganisms (6) and of BH4 in mammals (29). FH4 serves as a coenzyme for a variety of one-carbon transfer reactions (26), while BH4 functions as an essential reducing cofactor for NOSs, glyceryl ether mono-oxygenases, and mammalian aromatic amino acid hydroxylases (23, 29, 36, 37). Bacterial GTPCHs from Escherichia coli (44), Bacillus subtilis (10), Lactobacillus plantarum (17), and Serratia indica (21) are all known in folate biosynthetic pathways. Streptomyces tubercidicus (46) contains a GTP-8-formylhydrolase involved in the formation of pyrrolopyrimidine nucleoside antibiotics, an enzyme that cleaves the diazole ring but does not cyclize the product to produce pteridines as the final product. Instead, pyrrolopyrimidine is formed (11). GTPCHs involved in BH4 biosynthesis have been purified to homogeneity from human, mouse, and rat tissues (15, 20, 33), as well as from Drosophila melanogaster (41). To date, no GTPCH enzymes have been characterized in actinomycetes. In higher organisms, de novo BH4 biosynthesis has been extensively characterized, and the activity of GTPCH is regulated by a variety of mechanisms (16, 19, 37, 38, 42, 45). Dihydroneopterin triphosphate, the first product of GTPCH cleavage of GTP is ultimately converted to BH4 by the sequential action of 6-pyruvoyltetrahydropterin synthase (EC 4.6.1.10) and sepiapterin reductase (EC 1.1.1.153) (37) (Fig. ​(Fig.1).1). The process of biosynthesis of BH4 in bacteria is much less known, and the functions of BH4 in prokaryotes are not widely understood. Putative genes encoding BH4 biosynthetic proteins have been detected in the genomes of B. subtilis (22) and a Synechocystis sp. (18). A sepiapterin reductase producing l-threo-dihydrobiopterin was purified from Chlorobium tepidum (8). However, few studies on the characterization of GTPCHs involved in prokaryotic BH4 biosynthesis have been reported (25). FIG. 1. Pathways for BH4 biosynthesis and activity assay for GTPCH. PTPS, 6R-pyruvoyltetrahydropterin. This paper describes the purification and biochemical characterization of GTPCH from Nocardia sp. strain NRRL 5646, compares its properties with those of known GTPCHs, and probes its possible function in Nocardia NO synthesis.