The universally conserved N6-threonylcarbamoyladenosine (t6A) modification of tRNA is essential for translational fidelity. In bacteria, t6A biosynthesis starts with the TsaC/TsaC2-catalyzed synthesis of the intermediate threonylcarbamoyl adenylate (TC–AMP), followed by transfer of the threonylcarbamoyl (TC) moiety to adenine-37 of tRNA by the TC-transfer complex comprised of TsaB, TsaD and TsaE subunits and possessing an ATPase activity required for multi-turnover of the t6A cycle. We report a 2.5-Å crystal structure of the T. maritima TC-transfer complex (TmTsaB2D2E2) bound to Mg2+-ATP in the ATPase site, and substrate analog carboxy-AMP in the TC-transfer site. Site directed mutagenesis results show that residues in the conserved Switch I and Switch II motifs of TsaE mediate the ATP hydrolysis-driven reactivation/reset step of the t6A cycle. Further, SAXS analysis of the TmTsaB2D2-tRNA complex in solution reveals bound tRNA lodged in the TsaE binding cavity, confirming our previous biochemical data. Based on the crystal structure and molecular docking of TC–AMP and adenine-37 in the TC-transfer site, we propose a model for the mechanism of TC transfer by this universal biosynthetic system.

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.

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 several 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 (Ng) GCYH‐IB, and two high‐resolution crystal structures of the enzyme; one in complex with the reaction intermediate analog and a competitive inhibitor 8‐oxo‐GTP, and one with a TRIS molecule bound in the active site which mimics another reaction intermediate. Comparison of these GCYH‐IB crystal structures with the type IA enzyme bound to 8‐oxo‐GTP, reveals an inverted binding mode of the inhibitor's ribosyl moiety. Taken together with site‐directed mutagenesis data, suggests that the two enzymes utilize different strategies for catalysis. Additionally, the inhibitor interacts with a conserved active site residue Cys149 which 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. These structures and information were used to perform structure based drug design using a combination of in silico screening and de novo design methods to generate molecules that inhibit NgGCYH‐IB selectively over human GCYH‐IA. A few compounds named G1, G2 and G1‐triphosphate have been generated and currently being tested for inhibition.Support or Funding InformationThis project was supported by NSF grant CHE‐1309323 to D. Iwata‐Reuyl and M.A. Swairjo,NIH grant GM110588 to M.A. Swairjo and D. Iwata‐Reuyl, NIH grant GM70641 to D. Iwata‐Reuyl, andan intramural grant from Western University of Health Sciences to M.A. Swairjo.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

GTP cyclohydrolase I (GCYH‐I) catalyzes the first step in the de novo biosynthesis pathway to folic acid in bacteria, biopterin synthesis in mammals, and the biosynthesis of 7‐deazaguanine modified nucleosides of tRNA in bacteria and archaea. The type IB GTP cyclohydrolase (GCYH‐IB) is a prokaryotic‐specific, essential enzyme found in several pathogens, including N. gonorrhoeae, and is structurally distinct from its human homologue GCYH‐IA involved in biopterin biosynthesis and thus has been proposed as a novel antibacterial drug target. Comparison of the crystal structures of the two enzymes bound to the substrate analog inhibitor 8‐oxo‐GTP shows that the active site of GCYH‐IB is larger and imposes a different conformation of the inhibitor. Based on this structural information, we designed, synthesized and tested a small set of 8‐oxo‐G derivatives with ether linkages expected to occupy water‐filled pockets present only in the active site of GCYH‐IB. The half maximal inhibitory concentrations, measured using standard GCYH‐I activity assays, are indicative of modest potency of these compounds. However, the most potent of these compounds, G3, is selective for GCYH‐IB. In silico molecular modeling of G3 in the active sites of both enzymes supports the observed preferential inhibition of the N. gonorrhoeaea enzyme by G3 on the grounds that it is too large to be accommodated by the active site of human GCYH‐IA. These results support the premise that potent and selective inhibitors of GCYH‐IB could constitute a new class of small molecule antibiotics. Support or Funding Information NIGMS grant GM110588 and GM132254 to M.A.S., and The California Metabolic Research Foundation (SDSU).

GTP cyclohydrolase (GCYH-I) is an enzyme in the folate biosynthesis pathway that has not been previously exploited as an antibiotic target, although several pathogens including N. gonorrhoeae use a form of the enzyme GCYH-IB that is structurally distinct from the human homologue GCYH-IA. A comparison of the crystal structures of GCYH-IA and -IB with the nM inhibitor 8oxo-GTP bound shows that the active site of GCYH-IB is larger and differently shaped. Based on this structural information, we designed and synthesized a small set of 8-oxo-G derivatives with ether linkages at O 6 and O 8 expected to displace water molecules from the expanded active site of GCYH-IB. The most potent of these compounds, G3, is selective for GCYH-IB, supporting the premise that potent and selective inhibitors of GCYH-IB could constitute a new class of small molecule antibiotics.