Sequence analysis and overexpression of the Zymomonas mobilis tgt gene encoding tRNA-guanine transglycosylase: purification and biochemical characterization of the enzyme

Abstract

. We showed that this gene is able to complement the tgt mutation in E. coli SJ1505, and we determined its complete sequence. Four start codons were possible for this gene, resulting in proteins of 386 to 399 amino acids (M r , 42,800 to 44,300) showing 60.4% sequence identity with Tgt from E. coli. The smallest of the four possible reading frames, which was still extended at its 5 end compared with the E. coli tgt gene, was overexpressed in E. coli. The gene product was purified to homogeneity and was biochemically characterized. The kinetical parameters were virtually identical to those published for the E. coli enzyme. In contrast to E. coli Tgt, which is reported to be a homotrimer, Z. mobilis Tgt was found to be a monomer according to gel filtration. In this study, it was shown that the formation of homotrimers by the E. coli enzyme is readily reversible and is dependent on protein concentration. Asn in most eucaryotes and eubacteria. Since Q is a nutrient factor for eucaryotes, they contain Q as the free base as well, whereas eubacterial cells do not contain the free Q base. In eucaryotes, Q might be involved in many cellular processes such as differentiation (10, 32), the management of hypoxic stress (25, 26), the regulation of protein synthesis (25), the adaptive regulation of cell proliferation to the metabolic predisposition (18), and the modulation of signal transduction pathways (17). The hypermodified tRNA nucleoside queuosine [Q: 7-(((4,5-cis-dihydroxy-2-cyclopentene-1-yl)amino)methyl)-7-deaza-In eubacteria, the tRNAs are fully modified with respect to Q under all growth conditions. The exact function of Q in tRNA has not yet been clarified. Escherichia coli mutants deficient in Q biosynthesis die earlier during stationary phase in coculture with wild-type cells (22). They show a significantly higher rate of protein synthesis during outgrowth after nutritional starvation than wild-type cells (36). Thus, the Q in tRNAs seems to participate in the fine tuning of protein biosynthesis, as it is found for other tRNA modifications (2). There is recent evidence for a distinct function of Q in eubacterial tRNAs. A mutation giving rise to apathogenicity in the dysentery-causing bacterium Shigella flexneri was identified as a mutation in a Q biosynthesis gene. In this organism, Q deficiency leads to an inefficient translation of the virF mRNA, which contains the information for a positive regulator of further virulence genes. The mechanism of this phenomenon has not yet been clarified (9).Eubacteria synthesize Q de novo. Biosynthesis (see Fig. 1) starts outside the tRNA, with GTP being converted to 7-aminomethyl-7-deazaguanine (preQ 1 ) by one or several reactions that have not yet been characterized (15). preQ 1 is then inserted into the first position of the anticodon of the tRNA, replacing the genetically encoded guanine. The reaction is catalyzed by the enzyme tRNA-guanine transglycosylase (Tgt [EC 2.4.2.29]) (23). Further biosynthesis occurs at the tRNA level, where a substituted cyclopentyl group deriv...

Queuosine Formation in Eukaryotic tRNA Occurs via a Mitochondria-localized Heteromeric Transglycosylase

“…Conserved amino acids are shown in white on a dark gray background. The numbering refers to the Z. mobilis TGT sequence beginning at the fourth possible start codon GTG (Val), denoted by an arrow, as described previously (14). The secondary structure of the Z. mobilis TGT is shown above the sequences (11), whereas the predicted secondary structure elements of the QTRTD1 proteins, generated using the Porter program (27), are presented below the sequences.…”

“…Asp-102, which was originally ascribed the role of active site nucleophile, functions as a general acid/ base during catalysis (12,10). Although, the E. coli and Z. mobilis TGT enzymes are monomeric in solution (14), at high protein concentrations the E. coli enzyme can oligomerize (15), and structural data from the Z. mobilis TGT has shown the formation of a 2:1 complex with tRNA; a possible functional requirement for catalysis (10).…”

Brucella abortus Genes Identified following Constitutive Growth and Macrophage Infection

“…A second gene identified from the sequence of 36C2 was queuine tRNA ribotransferase. This enzyme is involved in the biosynthesis of the hypermodified tRNA nucleoside queuosine (Q) (33). Lastly, clone 33F5 exhibited homology with a gene encoding an ABC associated with mannopine transport (29).…”

Transglycosylation: A mechanism for RNA modification (and editing?)

“…For example, the eukaryotic TGT is a heterodimer that is comprised of a putative 60-kDa regulatory subunit and a 35-kDa catalytic subunit [13,14] while the eubacterial TGT has been considered to be monomeric [15,16] although a recent crystal structure shows the Zymomonas mobilis TGT to be a homodimer [17]. Additionally, the TGT from archaea is considerably larger, containing ca.…”

“…It is unclear if the trimeric structure is realized in vivo; however, it has been reported that the highly homologous TGT from Z. mobilis (ca. 60% similarity) exists as a monomer in the absence of tRNA [16]. Recently, studies in our laboratory have demonstrated that the E. coli TGT proceeds via a ping-pong kinetic mechanism whereby tRNA is the first substrate to bind to the enzyme [24].…”