The Characterization of tRNA Modifying Enzymes S-adenosylmethionine : tRNA Ribosyltransferase-isomerase (QueA) and a novel Type I GTP cyclohydrolase

Queuosine is a hypermodified nucleoside located in the wobble position of bacterial and eukaryotic tRNAs coding for Asp, Tyr, His and Asn. The biosynthesis involves the participation of S-adenosyl-methionine:tRNA ribosyltransferase-isomerase (QueA) and a GTP Cyclohydrolase-I. QueA catalyzes the transfer and isomerization of the ribosyl moiety from AdoMet to preQ1 modified tRNA. Substrate analogs of AdoMet were used to elucidate important substrate-enzyme interactions and to test key steps in the proposed chemical mechanism. Replacing AdoMet with SeAdoMet had little effect upon substrate binding but exhibited 30-fold reduction in kcat, consistent with deprotonation at C-5\u27 as the first catalytic step. 7-deazaAdoMet failed to function as a substrate of QueA, but exhibited a Ki that was only slightly higher than the K m for AdoMet, suggesting that N-7 is critical for catalysis but not substrate binding. Neither the 2\u27- or 3\u27-deoxyAdoMet exhibited activity with QueA, however both analogs had a Ki of only 2-fold higher than the Km of AdoMet. Reported here is the identification and characterization of the COG1469 protein family as a novel Type-I GTP cyclohydrolase (GCYH) that catalyzes the conversion of GTP to 7,8-dihydroneopterin-triphosphate in ∼20% of bacteria and most archaea. The COG1469 proteins and the genes that encode them were renamed GCYH-IB and folE-2, respectively, whereas the canonical cyclohydrolase, was renamed GCYH-IA. B. subtilis and N. gonorrhoeae GCYH-IB are homotrimers that were maximally active in the presence of manganese. GDP also functioned as a substrate; however the removal of the γ-phosphate resulted in a ∼30-fold decrease in kcat/Km. Inhibition analysis with N. gonorrhoeae GCYH-IB demonstrated that 8-oxoGTP functioned as a potent inhibitor with a K145-fold lower than the Km for GTP. Although the 7-deazaGTP did not function as a substrate for GCYH-IB, it exhibited a Ki 7-fold higher than the Km for GTP. The Ki for 2\u27-deoxyGTP was 24-fold higher than the Km of GTP. H245 of N. gonorrhoeae GCYH-IB was believed to function analogous to that of H179 of E. coli FolE which facilitates the elimination of C-8 from GTP. However, H245 mutants were still able to catalyze the elimination of C-8, suggesting that H245 is not involved in the deformylation of GTP

Functional Promiscuity of the COG0720 Family

The biosynthesis of GTP derived metabolites such as tetrahydrofolate (THF), biopterin (BH4), and the modified tRNA nucleosides queuosine (Q) and archaeosine (G+) relies on several enzymes of the Tunnel-fold superfamily. A subset of these proteins include the 6-pyruvoyl-tetrahydropterin (PTPS-II), PTPS-III, and PTPS-I homologs, all members of the COG0720 family, that have been previously shown to transform 7,8-dihydroneopterin triphosphate (H2NTP) into different products. PTPS-II catalyzes the formation of 6-pyruvoyltetrahydropterin in the BH4 pathway. PTPS-III catalyzes the formation of 6-hydroxylmethyl-7,8-dihydropterin in the THF pathway. PTPS-I catalyzes the formation of 6-carboxy-5,6,7,8-tetrahydropterin in the Q pathway. Genes of these three enzyme families are often misannotated as they are difficult to differentiate by sequence similarity alone. Using a combination of physical clustering, signature motif, and phylogenetic codistribution analyses, in vivo complementation studies, and in vitro enzymatic assays, a complete reannotation of the COG0720 family was performed in prokaryotes. Notably, this work identified and experimentally validated dual function PTPS-I/III enzymes involved in both THF and Q biosynthesis. Both in vivo and in vitro analyses showed that the PTPS-I family could tolerate a translation of the active site cysteine and was inherently promiscuous, catalyzing different reactions on the same substrate, or the same reaction on different substrates. Finally, the analysis and experimental validation of several archaeal COG0720 members confirmed the role of PTPS-I in archaeosine biosynthesis, and resulted in the identification PTPS-III enzymes with variant signature sequences in Sulfolobus species. This study reveals an expanded versatility of the COG0720 family members and illustrates that for certain protein families, extensive comparative genomic analysis beyond homology is required to correctly predict function

Spectroscopic Studies of Cu2+ and Zn2+ Binding to Prodigiosin Analogs

Prodigiosins are a family of secondary metabolites that were first isolated from the bacterium Serratia marascens. These natural compounds are red pigmented and characterized by a tri-pyrrole skeleton with a C-4 methoxy group. They have been reported to have good biological properties that include anticancer, antimalarial, antimicrobial and immunosuppressive activities. We have synthesized analogs of the natural prodigiosins (prodiginines) to produce a library of biologically active compounds which have improved biological activity and reduced cytotoxicity in human cells. In this work we studied the interaction between prodiginines and Cu2+ and Zn2+ using UV and Mass Spectroscopy techniques. Early results show that our prodigiosin analogs have good metal binding properties with dissociation constants (Kd) in the micromolar range. Understanding metal binding activities of prodiginines can be key in understanding their pharmacological action. With some drug-metal complexes already in use for the management of conditions such as cancer, diabetes, ulcers and rheumatoid arthritis there\u27s increased interest in this area

A class of hydrazones are active against non-replicating Mycobacterium tuberculosis.

There is an urgent need for the development of shorter, simpler and more tolerable drugs to treat antibiotic tolerant populations of Mycobacterium tuberculosis. We previously identified a series of hydrazones active against M. tuberculosis. We selected five representative compounds for further analysis. All compounds were active against non-replicating M. tuberculosis, with two compounds demonstrating greater activity under hypoxic conditions than aerobic culture. Compounds had bactericidal activity with MBC/MIC of < 4 and demonstrated an inoculum-dependent effect against aerobically replicating bacteria. Bacterial kill kinetics demonstrated a faster rate of kill against non-replicating bacilli generated by nutrient starvation. Compounds had limited activity against other bacterial species. In conclusion, we have demonstrated that hydrazones have some attractive properties in terms of their anti-tubercular activity

Mechanism and Catalytic Strategy of the Prokaryotic Specific GTP Cyclohydrolase IB

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

Functional Modular Dissection of DEBS1-TE Changes Triketide Lactone Ratios and Provides Insight into Acyl Group Loading, Hydrolysis, and ACP Transfer

The DEBS1-TE fusion protein is comprised of the loading module, the first two extension modules, and the terminal TE domain of the <i>Saccharopolyspora erythraea</i> 6-deoxyerythronolide B synthase. DEBS1-TE produces triketide lactones that differ on the basis of the starter unit selected by the loading module. Typical fermentations with plasmid-based expression of DEBS1-TE produce a 6:1 ratio of propionate to isobutyrate-derived triketide lactones. Functional dissection of the loading module from the remainder of DEBS1-TE results in 50% lower titers of triketide lactone and a dramatic shift in the production to a 1:4 ratio of propionate to isobutyrate-derived products. A series of radiolabeling studies of the loading module has shown that transfer from the AT to the ACP occurs much faster for propionate than for isobutyrate. However, the equilibrium occupancy of the AT favors isobutyrate such that propionate is outcompeted for ACP occupancy. Thus, propionyl-ACP is the kinetic product, while isobutyryl-ACP is the thermodynamic product. A slowed transfer from the loading domain ACP to first-extension module KS due to functional dissection of DEBS1-TE allows this isobutyryl-ACP-favored equilibrium to be realized and likely accounts for the observed shift in triketide lactone products