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.
The interfacial active site of the type IB GTP cyclohydrolase from Neisseria gonorrhoeae (grey and green ribbons), in complex with zinc (magenta ball) and the reaction intermediate analog and potent inhibitor 8-oxo-GTP (stick model). The structure sheds light on the complex and unique catalytic strategy of this potential antibacterial target, and offers a starting point for the design of specific inhibitors against the enzyme. For more information, please see study by Paranagama et al. in this issue, pages 1017–1039. Image provided by Manal Swairjo.
Mechanism and catalytic strategy of the prokaryotic-specific GTP cyclohydrolase-IB
Naduni Paranagama, Shilah A. Bonnett, Jonathan Alvarez, Amit Luthra, Boguslaw Stec, Andrew Gustafson, Dirk Iwata-Reuyl, Manal A. Swairjo; Mechanism and catalytic strategy of the prokaryotic-specific GTP cyclohydrolase-IB. Biochem J 15 March 2017; 474 (6): 1017–1039. doi: https://doi.org/10.1042/BCJ20161025
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