PMID:12559918

Citation	Rebelo, J, Auerbach, G, Bader, G, Bracher, A, Nar, H, Hösl, C, Schramek, N, Kaiser, J, Bacher, A, Huber, R and Fischer, M (2003) Biosynthesis of pteridines. Reaction mechanism of GTP cyclohydrolase I. J. Mol. Biol. 326:503-16
Abstract	GTP cyclohydrolase I catalyses the hydrolytic release of formate from GTP followed by cyclization to dihydroneopterin triphosphate. The enzymes from bacteria and animals are homodecamers containing one zinc ion per subunit. Replacement of Cys110, Cys181, His112 or His113 of the enzyme from Escherichia coli by serine affords catalytically inactive mutant proteins with reduced capacity to bind zinc. These mutant proteins are unable to convert GTP or the committed reaction intermediate, 2-amino-5-formylamino-6-(beta-ribosylamino)-4(3H)-pyrimidinone 5'-triphosphate, to dihydroneopterin triphosphate. The crystal structures of GTP complexes of the His113Ser, His112Ser and Cys181Ser mutant proteins determined at resolutions of 2.5A, 2.8A and 3.2A, respectively, revealed the conformation of substrate GTP in the active site cavity. The carboxylic group of the highly conserved residue Glu152 anchors the substrate GTP, by hydrogen bonding to N-3 and to the position 2 amino group. Several basic amino acid residues interact with the triphosphate moiety of the substrate. The structure of the His112Ser mutant in complex with an undefined mixture of nucleotides determined at a resolution of 2.1A afforded additional details of the peptide folding. Comparison between the wild-type and mutant enzyme structures indicates that the catalytically active zinc ion is directly coordinated to Cys110, Cys181 and His113. Moreover, the zinc ion is complexed to a water molecule, which is in close hydrogen bond contact to His112. In close analogy to zinc proteases, the zinc-coordinated water molecule is suggested to attack C-8 of the substrate affording a zinc-bound 8R hydrate of GTP. Opening of the hydrated imidazole ring affords a formamide derivative, which remains coordinated to zinc. The subsequent hydrolysis of the formamide motif has an absolute requirement for zinc ion catalysis. The hydrolysis of the formamide bond shows close mechanistic similarity with peptide hydrolysis by zinc proteases.
Links	PubMed
Keywords	Amino Acid Sequence; Animals; Binding Sites; Catalysis; Crystallization; Crystallography, X-Ray; Escherichia coli/enzymology; Escherichia coli/genetics; Formamides/chemistry; Formic Acids/metabolism; GTP Cyclohydrolase/chemistry; GTP Cyclohydrolase/genetics; GTP Cyclohydrolase/metabolism; Guanosine Triphosphate/metabolism; Hydrogen Bonding; Hydrolysis; Kinetics; Models, Molecular; Molecular Sequence Data; Mutagenesis, Site-Directed; Mutation; Neopterin/analogs & derivatives; Protein Conformation; Pteridines/metabolism; Stereoisomerism; Zinc
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Annotations

Gene product	Qualifier	GO ID	GO term name	Evidence Code	with/from	Aspect	Notes	Status
FolE		GO:0008270	zinc ion binding	IDA: Inferred from Direct Assay		F	The Zn content of purified FolE is 0.9 mol/mol (Table 1). Purified FolE in 1 M HCl was heated at 96°C for 2 hours; the amount of Zn released was then determined by atomic absorption spectrometry.	complete
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References

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