Active site topology and reaction mechanism of GTP cyclohydrolase I.

Abstract

(5), compound 4 is the biosynthetic precursor of tetrahydrobiopterin, which is involved in the formation of catecholamines and nitric oxide (6, 7). Tetrahydrobiopterin is also involved in the stimulation of T lymphocytes, although the details are still incompletely understood (8).CYH was originally detected in bacteria (9). More recently, the primary structure of CYH from a wide variety of organisms has been determined (10-16). The evolution of the protein has been relatively conservative. Thus, the C-terminal 120 residues of Escherichia coli and human enzymes are 60% identical. The crystal structure of E. coli CYH has recently been solved by single isomorphous replacement and averaging techniquesThe publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. (17). The knowledge of the crystal packing arrangement obtained by electron microscopy (18, 19) was helpful in the initial stages of structure determination. The enzyme complex, a decamer consisting of a pentamer of tightly associated dimers, is doughnut shaped with dimensions of 65 x 100 A.One CYH subunit folds into a a+f structure with a predominantly helical N terminus (Fig. 2). The N-terminal helix hl (residues 5-18) is followed by a helix pair, composed of helix h2 (residues 32-49) and helix h3 (residues 62-72), which are remote from the compact C-terminal domain of the molecule (residues 95-217). This domain has a central fourstranded antiparallel 13-sheet that is flanked on both sides by a-helices. The folding topology of this domain is identical to that of a subunit of the second enzyme in tetrahydrobiopterin biosynthesis pathway, 6-pyruvoyltetrahydropterin synthase (20).The association of two CYH monomers to dimers is driven by the formation of a four-helix bundle by helices h2 and h3 of each monomer. Apart from a large hydrophobic surface buried by this interaction, numerous hydrogen bonds and salt bridges serve to stabilize the dimer. The decamer is formed by a fivefold symmetric arrangement of the dimers. Its most striking structural feature is an unprecedented 20-stranded antiparallel ,B-barrel (Fig. 2). This paper describes the crystal structure of a complex of CYH with dGTP, the substrate analog.** By using the inforAbbreviation: CYH, GTP cyclohydrolase I.

GTP cyclohydrolase I utilizes metal‐free GTP as its substrate

“…Our data showed that the Mg-GTP complex and Mg 2+ affected the enzyme activity very little at the concentrations tested here. The structure of the active centre for the E. coli GCH binding to dGTP also supports our findings, because Mg 2+ assistance for binding to the GTP substrate was neither realized nor necessary [14].…”

“…However, the apo-enzyme is probably unstable, because the addition of Zn 2+ after refolding was less effective than that during refolding (Table 1). Auerbach et al previously suggested that the absence of Zn 2+ in the active centre of the GCH enzyme caused enzymatic inactivation by disulfide formation between the cysteine residues [14] which normally form the Zn 2+ binding site.…”

“…By crystallographic analysis using purified Escherichia coli enzyme [14], an N-terminally truncated form of the recombinant human enzyme [12], and a stimulatory complex of rat GCH and GFRP induced by phenylalanine [15], Zn 2+ was shown to be bound to the active centre of the homodecameric GCH enzyme. As for Ca 2+ , mutations of the recombinant rat enzyme in an EF-hand-like motif, which is absent in bacteria, inhibited both the binding of Ca 2+ to the enzyme and enzyme activity [13].…”

Folate synthesis in plants: The first step of the pterin branch is mediated by a unique bimodular GTP cyclohydrolase I

“…1). GCHI has been cloned and characterized from Escherichia coli, yeast, and mammals (in mammals it participates in tetrahydrobiopterin synthesis), and the crystal structures of the E. coli and human enzymes have been solved (11)(12)(13). All the cloned GCHI polypeptides have 235 Ϯ 15 residues and a molecular mass of 26 Ϯ 2 kDa.…”

“…E. coli and human GCHIs are homodecamers comprising five tightly associated dimers. There are 10 active sites per decamer, each located at the interface of three subunits and each with a catalytically essential zinc ion bound by histidine and cysteine side chains (11,12). Plant GCHIs have not been cloned, but the enzyme has been isolated from spinach leaves and partially characterized (14).…”