SERINE-90 IS REQUIRED FOR ENZYMATIC-ACTIVITY BY TRANSFER-RNA-GUANINE TRANSGLYCOSYLASE FROM ESCHERICHIA-COLI

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 preQ(1) 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. Determination of Michaelis-Menten parameters showed that the mutation of S90 to C yielded a 40-fold reduction in V-max/K-M for tRNA and a 5-fold reduction in V-max/K-M for guanine, while V-max was relatively unchanged (ca. 25% lower). Most of the change in V-max/K-M for tRNA came from a 30-fold increase in K-M. The enzymic activity of the S90A mutant was too low to determine V-max and K-M; however, the V-max/K-M for each substrate was reduced by 4 orders of magnitude compared to the wild type. These results indicate that serine 90 is performing a critical role in the TGT reaction.