Serine 90 Is Required for Enzymic Activity by tRNA-Guanine Transglycosylase from Escherichia coli

Abstract

An Escherichia coli mutant described by Noguchi et al. [Noguchi, S., et al. (1982) J. Biol. Chem. 275, 6544-6550] contains tRNA lacking the hypermodified wobble nucleoside queuosine (Q) due to an inactive tRNA-guanine transglycosylase (TGT). TGT catalyzes the posttranscriptional base exchange of the Q precursor preQ1 with the genetically encoded guanine in tRNA(Asp,Asn,His,Tyr). The mutant tgt gene was cloned and sequenced; it contained a single point mutation resulting in the change of serine 90 to phenylalanine. Overexpression of the mutant gene yielded TGT(S90F) that showed a reduced solubility and did not purify in the same fashion as the wild-type enzyme. TGT(S90F) has no detectable enzymic activity. To determine whether serine 90 performs a catalytic role in the TGT reaction or whether the loss of activity was caused solely by a conformational change of the enzyme, we used site-specific mutagenesis to construct serine-to-alanine (S90A) and serine-to-cysteine (S90C) mutants. Both S90A and S90C mutants were purified in a manner identical to that used for the wild-type enzyme. SDS-PAGE of dimethyl suberimidate-cross-linked mutants showed a pattern identical to that of the wild-type TGT, indicative of a trimeric quaternary structure. Native PAGE of wild-type and mutant TGTs in the absence and presence of substrate tRNA exhibited band shifts indicating that both mutants retain the ability to bind tRNA.(ABSTRACT TRUNCATED AT 250 WORDS)

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

“…Recently, we have reported that Ser-90 of the E. coli Tgt enzyme is essential for efficient catalysis (27). Mutated Tgts from E. coli with Ser-903Phe or Ser903Ala are inactive, while the Tgt with Ser-903Cys shows a reduced activity.…”

“…By gel filtration, the apparent molecular mass of Z. mobilis Tgt was determined to be 55 kDa, suggesting a monomeric form. In contrast, it is known by cross-linking experiments and native PAGE that E. coli Tgt forms a homotrimer (5,6,13,27). Therefore, we examined the native molecular mass of the E. coli enzyme by gel filtration.…”

“…The determination of Tgt activity was carried out with a reaction mixture containing 10 mM Tris-HCl (pH 7.5), 10 mM MgCl 2 , and 2 mM dithiothreitol, saturating concentrations of [ , and various amounts of protein sample in a total volume of 50 l. E. coli tRNA Tyr was prepared as described previously (27). The reaction mixtures were incubated at 37ЊC, and 10-l aliquots were taken at various time points.…”

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

“…Site-directed mutagenesis studies determined that the observed loss of activity was catalytically significant, and not merely the result of a conformational change brought about by the non-conservative mutation [23]. This finding implicated an important role for serine 90 in TGT catalysis.…”

“…The recombinant TGT from E. coli is a 43-kDa enzyme that exists as a homotrimer in solution but dissociates into a monomer upon tRNA binding [22,23]. 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.…”