Published In

Biochemical Journal

Document Type

Post-Print

Publication Date

3-1-2017

Subjects

Transfer RNA, Microbial metabolism, Metalloenzymes, Nucleosides, Post-translational modification

Abstract

GTP cyclohydrolase 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 GTP cyclohydrolase (GCYH-IB) is a prokaryotic-specific enzyme found in a number of pathogens. GCYH-IB is structurally distinct from the canonical type IA GTP cyclohydrolase 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-oxo-GTP, and one with a TRIS molecule bound in the active site and mimicking another reaction intermediate. Comparison with the type IA enzyme bound to 8-oxo-GTP 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.

Description

Published in final edited form as: Biochem J. ; 474(6): 1017–1039. doi:10.1042/BCJ20161025.

DOI

0.1042/BCJ20161025

Persistent Identifier

http://archives.pdx.edu/ds/psu/21870

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