Characterization of Bacillus caldotenax anthranilate synthase I produced in Escherichia coli and identification of its essential arginine residue by site-directed mutagenesis.
Anthranilate synthase I (ASI) of Bacillus caldotenax, a thermophilic bacterium, was purified from a plasmid-bearing Escherichia coli and characterized. The molecular weight determination under native and denaturing conditions revealed that it was a monomeric enzyme of M(r) = 54,000. The N-terminal amino acid sequence is the same as expected from DNA sequence of trpE except that the N-terminal methionine is lacking. All four cysteines in the molecule could be titrated with 5,5'-dithiobis (2-nitrobenzoic acid) in more than 8 M urea. The purified enzyme retained its full enzymatic activity after being heated at 60 degrees C. Six mutated genes for the ASI with histidine in place of each conserved arginine, Arg321, Arg353, Arg358, Arg416, Arg429, and Arg452, were prepared by site-directed mutagenesis. All the mutated genes except one, the gene encoding an ASI mutant with histidine in place of Arg452 (R452H ASI) complemented an E. coli (trpE). The mutated ASIs were purified and compared with the wild type ASI. No distinctive differences in enzymatic properties were found between the wild type and the enzymatically active mutated ASIs. R452H ASI was enzymatically inactive, though its conformation seemed to be unchanged after the substitution based on CD spectra and the SH titration curve.
Shiratsuchi
A
A
Mitsubishi-Kasei Institute of Life Sciences, Tokyo.
Sato
S
S
D000595:Amino Acid Sequence; D000878:Anthranilate Synthase / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification; D001120:Arginine / Q000032:analysis*; D001407:Bacillus / Q000201:enzymology*; D001483:Base Sequence; D002942:Circular Dichroism; D003001:Cloning, Molecular; D004269:DNA, Bacterial; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D015183:Restriction Mapping; D016415:Sequence Alignment
D004269:DNA, Bacterial; D001120:Arginine; D000878:Anthranilate Synthase