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.
Rühlmann
A
A
Max-Planck-Institut fur Experimentelle Medizin, Department of Chemistry, Göttingen, Germany. RHL@IFM.MH-Hannover.de
Cramer
F
F
Englisch
U
U
D000595:Amino Acid Sequence; D000818:Animals; D001419:Bacteria / Q000201:enzymology; D002852:Chromatography, Ion Exchange; D004926:Escherichia coli / Q000201:enzymology*; D006643:Histidine-tRNA Ligase / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D006801:Humans; D008715:Methionine; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017354:Point Mutation; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D016415:Sequence Alignment; D017384:Sequence Deletion; D014633:Valine
D011993:Recombinant Fusion Proteins; D008715:Methionine; D006643:Histidine-tRNA Ligase; D014633:Valine