CATALYTIC DEFECTS IN MUTANTS OF CLASS-II HISTIDYL-TRANSFER-RNA SYNTHETASE FROM SALMONELLA-TYPHIMURIUM PREVIOUSLY LINKED TO DECREASED CONTROL OF HISTIDINE BIOSYNTHESIS REGULATION

The expression of histidine biosynthetic genes in enteric bacteria is regulated by an attenuation mechanism in which the level of histidyl-t RNA serves as a key sensor of the intracellular histidine pool. Among the early observations that led to the formation of this model for Sal monella typhimurium were the identification of mutants in the gene (hi sS) encoding histidyl-tRNA synthetase. We report here the detailed bio chemical characterization of five of these S. typhimurium bradytrophic mutants isolated by selection for resistance to histidine analogs, in cluding identification of the deduced amino acid substitutions and det ermination of the resulting effects on the kinetics of adenylation and aminoacylation. Using the crystal structure of the closely related Es cherichia coil histidyl-tRNA synthetase (HisRS) as a guide, two mutant s were mapped to a highly conserved proline residue in motif 2 (P117S, P117Q), and were correlated with a fivefold decrease in the k(cat) fo r the pyrophosphate exchange reaction, as well as a tenfold increase i n the K-m for tRNA in the aminoacylation reaction. Another mutant subs titution (A302T) mapped to a residue adjacent to the histidine binding pocket, leading to a tenfold increase in K-m for histidine in the pyr ophosphate exchange reaction. The remaining two mutants (S167F, N254T) substitute residues in or directly adjacent to the hinge region, whic h joins the insertion domain between motif 2 and motif 3 to the cataly tic core, and cause the K-m for tRNA to increase four- to tenfold. The kinetic analysis of these mutants establishes a direct link between c ritical interactions within the active site of HisRS and regulation of histidine biosynthesis, and provides further evidence for the importa nce of local conformational changes during the catalytic cycle. (C) 19 98 Academic Press.