title: Mechanism and catalytic strategy of the prokaryotic-specific GTP cyclohydrolase-IB.
abstract: 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 ofNeisseria gonorrhoeaeGCYH-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 theS-nitrosothiol in catalysis.
authors.affiliation: Department of Chemistry and Biochemistry, San Diego State University, 5500 Campanile Drive, San Diego, CA 92182, U.S.A.
authors.forename: Naduni
authors.initials: N
authors.lastname: Paranagama
authors.affiliation: Department of Chemistry, Portland State University, PO Box 751, Portland, OR 97207, U.S.A.
authors.forename: Shilah A
authors.initials: SA
authors.lastname: Bonnett
authors.affiliation: The Graduate College of Biomedical Sciences, Western University of Health Sciences, 309 E. Second Street, Pomona, CA 91766, U.S.A.
authors.forename: Jonathan
authors.initials: J
authors.lastname: Alvarez
authors.affiliation: Department of Chemistry and Biochemistry, San Diego State University, 5500 Campanile Drive, San Diego, CA 92182, U.S.A.
authors.forename: Amit
authors.initials: A
authors.lastname: Luthra
authors.affiliation: Department of Chemistry and Biochemistry, San Diego State University, 5500 Campanile Drive, San Diego, CA 92182, U.S.A.
authors.forename: Boguslaw
authors.initials: B
authors.lastname: Stec
authors.affiliation: Department of Chemistry, Portland State University, PO Box 751, Portland, OR 97207, U.S.A.
authors.forename: Andrew
authors.initials: A
authors.lastname: Gustafson
authors.affiliation: Department of Chemistry, Portland State University, PO Box 751, Portland, OR 97207, U.S.A. iwatard@pdx.edu mswairjo@mail.sdsu.edu.
authors.forename: Dirk
authors.initials: D
authors.lastname: Iwata-Reuyl
authors.affiliation: Department of Chemistry and Biochemistry, San Diego State University, 5500 Campanile Drive, San Diego, CA 92182, U.S.A. iwatard@pdx.edu mswairjo@mail.sdsu.edu.
authors.forename: Manal A
authors.initials: MA
authors.lastname: Swairjo
mesh_terms: D001426:Bacterial Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D020134:Catalytic Domain; D003001:Cloning, Molecular; D018360:Crystallography, X-Ray; D004791:Enzyme Inhibitors / Q000737:chemistry; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D006136:GTP Cyclohydrolase / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D006160:Guanosine Triphosphate / Q000031:analogs & derivatives* / Q000737:chemistry; D007700:Kinetics; D008958:Models, Molecular; D009154:Mutation; D009344:Neisseria gonorrhoeae / Q000737:chemistry* / Q000201:enzymology; D011485:Protein Binding; D000072756:Protein Conformation, alpha-Helical; D000072757:Protein Conformation, beta-Strand; D054730:Protein Interaction Domains and Motifs; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D026403:S-Nitrosothiols / Q000737:chemistry; D013379:Substrate Specificity; D014325:Tromethamine / Q000737:chemistry*
chemical_list: D001426:Bacterial Proteins; D004791:Enzyme Inhibitors; D011994:Recombinant Proteins; D026403:S-Nitrosothiols; D014325:Tromethamine; C057674:8-hydroxyguanosine triphosphate; D006160:Guanosine Triphosphate; D006136:GTP Cyclohydrolase
keywords: 7-deazaguanosine; bacterial metabolism; metalloenzyme; modified nucleoside; post-translational modification (PTM); transfer RNA (tRNA)