PT - JOURNAL ARTICLE AU - Paranagama, Naduni AU - Bonnett, Shilah A. AU - Alvarez, Jonathan AU - Luthra, Amit AU - Stec, Boguslaw AU - Gustafson, Andrew AU - Iwata-Reuyl, Dirk AU - Swairjo, Manal A. TI - Mechanism and catalytic strategy of the prokaryotic-specific GTP cyclohydrolase-IB DP - 2017 Mar 15 TA - Biochemical Journal PG - 1017--1039 VI - 474 IP - 6 4099 - http://www.biochemj.org/content/474/6/1017.short 4100 - http://www.biochemj.org/content/474/6/1017.full SO - Biochem J2017 Mar 15; 474 AB - Accepted Manuscript online January 26, 2017.Guanosine 5′-triphosphate (GTP) cyclohydrolase-I (GCYH-I) catalyzes the first step in folic acid biosynthesis in bacteria and plants, biopterin biosynthesis in mammals, and the biosynthesis of 7-deazaguanosine-modified tRNA nucleosides in bacteria and archaea. The type IB GCYH (GCYH-IB) is a prokaryotic-specific enzyme found in many pathogens. GCYH-IB is structurally distinct from the canonical type IA GCYH involved in biopterin biosynthesis in humans and animals, and thus is of interest as a potential antibacterial drug target. We report kinetic and inhibition data of Neisseria gonorrhoeae GCYH-IB and two high-resolution crystal structures of the enzyme; one in complex with the reaction intermediate analog and competitive inhibitor 8-oxoguanosine 5′-triphosphate (8-oxo-GTP), and one with a tris(hydroxymethyl)aminomethane molecule bound in the active site and mimicking another reaction intermediate. Comparison with the type IA enzyme bound to 8-oxo-GTP (guanosine 5′-triphosphate) reveals an inverted mode of binding of the inhibitor ribosyl moiety and, together with site-directed mutagenesis data, shows that the two enzymes utilize different strategies for catalysis. Notably, the inhibitor interacts with a conserved active-site Cys149, and this residue is S-nitrosylated in the structures. This is the first structural characterization of a biologically S-nitrosylated bacterial protein. Mutagenesis and biochemical analyses demonstrate that Cys149 is essential for the cyclohydrolase reaction, and S-nitrosylation maintains enzyme activity, suggesting a potential role of the S-nitrosothiol in catalysis.2′-dGTP, 2′-deoxyguanosine triphosphate; 7-deaza-GTP, 7-deazaguanosine 5′-triphosphate; 8-oxo-GTP, 8-oxoguanosine 5′-triphosphate; βME, β-mercaptoethanol; BSA, bovine serum albumin; DTT, dl-dithiothreitol; GCYH-I, GTP cyclohydrolase I; GCYH-IA, Type IA GTP cyclohydrolase; GCYH-IB, Type IB GTP cyclohydrolase; GSH, reduced glutathione; GTP, guanosine 5′-triphosphate; H2NTP, 7,8-dihydroneopterin triphosphate; HENS, buffer containing 100 mM HEPES, 1 mM EDTA, 0.1 mM neocuproine, 1% (w/v) SDS, pH 7.4; HEPES, N-(2-hydroxyethyl)piperazine-N′-(2-ethanesulfonic acid); HPLC, high-pressure liquid chromatography; MWCO, molecular mass cut-off; Ni-NTA, Nickel-Nitrilotriacetic Acid; PVDF, Polyvinylidene difluoride; SDS–PAGE, sodium dodecyl sulfate–polyacrylamide gel electrophoresis; SNO, S-nitrosothiol; TBST, TRIS-buffered saline and Tween 20; T-fold, tunnel-fold; TRIS, tris(hydroxymethyl)aminomethane.