Isolation and analysis of mutated histidyl-tRNA synthetases from Escherichia coli

Amino terminally deleted and point-mutated histidyl-tRNA synthetases were purified from E. coli via betaGal fusion proteins. A hinge region proximal and distal to the factor Xa cleavage region was necessary to cut the betaGal-fusion proteins efficiently under very mild nondenaturing conditions. N-terminal addition of either methionine or valine to this enzyme (its starting N-formyl-methionine is in vivo post-translationally removed) or the deletion of 6 amino terminal amino acids decreased the specific aminoacylation activity 2- to 7-fold. Further N-terminal deletions of 10 or 17 amino acids caused significantly reduced aminoacylation (100-fold) and ATP/PPi exchange (10-fold) activities, and a reduced binding affinity for histidine. Removal of 18 or more amino acids from the N-terminus thereby removing residues from MOTIF 1 resulted in inactive histidyl-tRNA synthetase mutants. Two point mutations within the histidyl-adenylate binding pocket, R259Q and R259K, also blocked histidyl-tRNA synthetase activity without affecting histidine or ATP binding. The experiments shown identify a highly conserved N-terminal R/KG-patch in front of MOTIF 1 as well as R259 as vital for full enzymatic activity.