Glu571 of PheT plays a pivotal role in the thermal stability of Escherichia coli PheRS enzyme.

ABSTRACT

As of date the two temperature sensitive mutations isolated in pheST operon include pheS5 (G293 →A293 ) and pheT354. Recently, we reported that G673 of pheS defines a hot spot for intragenic suppressors of pheS5. In this investigation, in 13 independent experiments, a collection of temperature sensitive mutants were isolated by localized mutagenesis. Complementation using clones bearing pheS+ , pheT+ , and pheS+ T+ indicated that 34 mutants could harbor lesion(s) in pheS and four could be in pheT and one mutant might be a double mutant. Surprisingly, all the 34 pheS mutants harbored the very same (G293 →A293 ) transition mutation as present in the classical pheS5 mutant. Most unexpectedly, the four pheT mutants isolated harbored the same G1711 →A1711 transition, a mutation which is hitherto unreported. Since all the four pheT mutants were defined by the same G1711 →A1711 base change, we believe that getting other mutations could be hard hitting and therefore it is proposed that G1711 itself could be a "hot spot" for emergence of Ts mutations in pheT and similarly G293 itself could be a "hot spot" for Ts lesions in pheS. These results clearly imply a vital role for Glutamic acid571 (Glu571 ) of PheT and reinforce criticality of Glycine98 (Gly98 ) of PheS in the thermal stability of PheRS enzyme.