Biochemical and structural studies of TGT and MiaA: Key enzymes involved in two types of hypermodifications

My PhD research studies two types of hypermodifications in tRNAs, which are described in part I and II respectively.;Part I. Hypermodification of base guanine at position 34. The first base modification is the replacement of the base guanine (G) by queuine (Q) at the wobble position 34 in tRNAs and the tRNA-guanine transglycosylase (TGT) is the key enzyme responsible for the catalysis. We successfully trapped the covalent intermediate during the catalysis and determined the crystal structure of TGT-RNA-9dzG ternary complex at a resolution of 2.9A. The crystal structure of the covalent intermediate provides us with valuable information about the enzymatic mechanism and molecular recognition, which was confirmed by site-directed mutagenesis followed by enzymatic assay. We then carried out chemical reactions in the covalent intermediate in the crystalline form by incubating the crystal with excess preQ1. Crystal structure of the reaction products clearly showed preQ1 bound in the substrate pocket. According to our structural and mutational studies, a mechanism for the TGT catalysis was proposed. Follow-up studies focused on the crystallization of TGT-tRNA-9dzG ternary complex in order to study the in vivo catalysis of TGT. This attempt was largely unsuccessful.;Part II. Hypermodification of base adenosine at position 37. Hypermodification of A37 (position 3' adjacent to the anticodon region) starts with the formation of (isopentenyl)-adenosine (i6A) and in E. coli, the first step is catalyzed by the enzyme MiaA. Through BLAST search, a few protein sequences highly homologous to MiaA were identified. These protein-encoding genes were cloned, over-expressed and purified. Enzymatic assays were carried out to test the putative enzymes but no activities were detected. The failure to detect activities might result from incorrect assay conditions or substrates and more data is yet to be acquired on this aspect. Crystals of the P. aeruginosa protein were obtained and its structure was determined at 2.2 A. Although the electron density of a segment of the protein was not observed, the partial structure nevertheless shows a central channel composed of positive charged residues. A hypothesis of the MiaA catalytic mechanism is proposed.