Evolution of eukaryal tRNA-guanine transglycosylase: insight gained from the heterocyclic substrate recognition by the wild-type and mutant human and Escherichia coli tRNA-guanine transglycosylases

The enzyme tRNA-guanine transglycosylase (TGT) is involved in the queuosine modification of tRNAs in eukarya and eubacteria and in the archaeosine modification of tRNAs in archaea. However, the different classes of TGTs utilize different heterocyclic substrates (and tRNA in the case of archaea). Based on the X-ray structural analyses, an earlier study [Stengl et al. (2005) Mechanism and substrate specificity of tRNA-guanine transglycosylases (TGTs): tRNA-modifying enzymes from the three different kingdoms of life share a common catalytic mechanism. Chembiochem, 6, 1926–1939] has made a compelling case for the divergent evolution of the eubacterial and archaeal TGTs. The X-ray structure of the eukaryal class of TGTs is not known. We performed sequence homology and phylogenetic analyses, and carried out enzyme kinetics studies with the wild-type and mutant TGTs from Escherichia coli and human using various heterocyclic substrates that we synthesized. Observations with the Cys145Val (E. coli) and the corresponding Val161Cys (human) TGTs are consistent with the idea that the Cys145 evolved in eubacterial TGTs to recognize preQ1 but not queuine, whereas the eukaryal equivalent, Val161, evolved for increased recognition of queuine and a concomitantly decreased recognition of preQ1. Both the phylogenetic and kinetic analyses support the conclusion that all TGTs have divergently evolved to specifically recognize their cognate heterocyclic substrates

Mechanistic studies of the reaction catalyzed by eubacterial tRNA -guanine transglycosylase.

The modified base queuine (Q) is found at the wobble position of tRNAs with anticodon sequences of GUN. In eubacteria a Q-precursor, preQ 1, is exchanged for guanine-34 by tRNA-guanine transglycosylase (TGT). Further modification at position 34 leads to Q-tRNA. Relatively little is known about the factors that influence the initiation and control of the TGT-catalyzed reaction. The kinetic mechanism of TGT was determined using initial rate kinetics and competitive inhibition studies. TGT was found to proceed through a ping-pong mechanism, with tRNA binding first. To facilitate our understanding of the chemical mechanism, the role of an active site cysteine (145 in E. coli) was investigated. A series of mutants was generated to probe the effects of charge and hydrogen-bonding potential at this position. The C145A and C145S mutants each showed a moderate (ca. 10-fold) increase in kcat, with corresponding increases in the KMs for both substrates. Alternatively, the aspartic acid mutant did not significantly alter the catalytic rate, but did cause 20- and 200-fold increases in the tRNA and guanine K Ms, respectively. To better understand the Significance of the kinetic studies, the pH-dependence of the TGT reaction was determined. The wild-type and mutant enzymes Showed bell-shaped pH profiles. Although the wild-type and mutant enzymes differ in pH optimum, this alone does not account for the altered activity seen with the mutants. Finally, the binding of both substrates was investigated. No difference in binding was observed between the wild-type and mutant TGTs for either G(34)-tRNA or preQ1(34)-tRNA. The binding of guanine was estimated through the use of a known competitive inhibitor (7-methylguanine). The Ki values for C145A and C145S were ca. 100- and 500-fold greater than WT, respectively. The Ki for C145D was beyond the limits of 7-methylguanine solubility. The results of this work support the hypothesis that Cys145 is involved in heterocycle binding and specificity. Both the rate enhancements, and the increases in KM for the C145A and C145S mutants are consistent with product (guanine) release being partially rate-limiting. The loss of the cysteine functionality weakens the interaction with guanine and allows faster dissociation of the base following bond cleavage.Ph.D.BiochemistryBiological SciencesMolecular biologyPharmacy sciencesPure SciencesUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/129511/2/3042075.pd

Crystal Structure Analysis and in Silico pKa Calculations Suggest Strong pKa Shifts of Ligands as Driving Force for High-Affinity Binding to TGT

A novel ligand series is presented to inhibit tRNA guanine transglycosylase TGT , a protein with a significant role in the pathogenicity mechanism of Shigella flexneri, the causative agent of Shigellosis. The enzyme exchanges guanine in the wobble position of tRNA Asn,Asp,His,Tyr against a modified base. To prevent the base exchange reaction, several series of inhibitors have already been designed, synthesized, and tested. One aim of previous studies was to address a hydrophobic pocket with different side chains attached to the parent skeletons. Disappointingly, no significant increase in binding affinity could be observed that could be explained by the disruption of a conserved water cluster. The ligand series examined in this study are based on the known scaffold lin benzoguanine. Different side chains were introduced leading to 2 amino lin benzoguanines, which address a different pocket of the protein and avoid disruption of the water cluster. With the introduction of an amino group in the 2 position, a dramatic increase in binding affinity can be experienced. To explain this significant gain in binding affinity, Poisson Boltzmann calculations were performed to explore pK a changes of ligand functional groups upon protein binding, they can differ significantly on going from aqueous solution to protein environment. For all complexes, a permanent protonation of the newly designed ligands is suggested, leading to a charge assisted hydrogen bond in the protein ligand complex. This increased strength in hydrogen bonding takes beneficial effect on binding affinity of the ligands, resulting in low nanomolar binders. Crystal structures and docking emphasize the importance of the newly created charge assisted hydrogen bond. A detailed analysis of the crystal structures in complex with substituted 2 amino lin benzoguanines indicate pronounced disorder of the attached side chains addressing the ribose 33 binding pocket. Docking suggests multiple orientations of these side chains. Obviously, an entropic advantage of the residual mobility experienced by these ligands in the bound state is beneficial and reveals an overall improved protein bindin

Enzymatic covalent labeling of RNA with RNA transglycosylation at guanosine (RNA-TAG)

Technologies for the labeling, detection, and manipulation of biomolecules have drastically improved our understanding of cell biology. As the myriad of functional roles for RNA in the cell are increasingly recognized, such tools to enable further investigation of RNA are the subject of much interest. RNA-TAG is an enzymatic method for site-specific, covalent labeling of RNA. This methodology makes use of a bacterial tRNA modifying enzyme, tRNA guanine transglycosylase, to incorporate modified substrate analogs into a target RNA, resulting in highly efficient and site-specific RNA labeling. In this chapter, we introduce the underlying principles of the RNA labeling reaction, discuss various applications of RNA-TAG, and present protocols for labeling specific RNA transcripts using this system