The in vitro conversion of chorismate to isochorismate catalyzed by the Escherichia coli entC gene product. Evidence that EntA does not contribute to isochorismate synthase activity.
The entC and entA genes, coding for the enzymes isochorismate synthase and 2,3-dihydro-2,3-dihydroxybenzoate dehydrogenase, respectively, were subcloned behind the T7 promoter in the expression plasmid pGEM3Z. Their protein products were overproduced and partially purified for in vitro analysis of the conversion of chorismate to isochorismate. Whereas previous genetic experiments suggested that the EntA enzyme has a role in this conversion, this study clearly indicates that EntC alone catalyzes the reaction. Addition of EntA had no effect on isochorismate synthase activity. As a result, the mutation (previously designated entC401) in strain AN191 was characterized by nucleotide sequence analysis. The lesion is a single base substitution in the entA gene, resulting in a glutamic acid-for-glycine substitution at the penultimate amino acid (residue 247) of the EntA enzyme. The mutant protein was partially purified and shown to be devoid of 2,3-dihydro-2,3-dihydroxybenzoate dehydrogenase activity, whereas the entC gene product from strain AN191 exhibited normal isochorismate synthase function. These results conflict with the earlier characterization of the entC401 mutation in a different genetic background. The data presented herein establish that the EntA protein does not contribute to isochorismate synthase activity and that the mutant strain that led to this suggestion harbors a defective allele of entA rather than entC.
Tummuru
M K
MK
Department of Microbiology, School of Medicine, University of Missouri, Columbia 65212.
Brickman
T J
TJ
McIntosh
M A
MA
D001483:Base Sequence; D002827:Chorismic Acid / Q000378:metabolism*; D003509:Cyclohexanecarboxylic Acids / Q000378:metabolism*; D053138:Cyclohexenes; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics*; D029968:Escherichia coli Proteins; D005798:Genes, Bacterial; D005838:Genotype; D019751:Intramolecular Transferases; D007535:Isomerases / Q000235:genetics* / Q000378:metabolism; D007536:Isomerism; D007700:Kinetics; D008969:Molecular Sequence Data; D010088:Oxidoreductases / Q000235:genetics*; D044925:Oxidoreductases Acting on CH-CH Group Donors; D011401:Promoter Regions, Genetic; D015183:Restriction Mapping; D013045:Species Specificity
D003509:Cyclohexanecarboxylic Acids; D053138:Cyclohexenes; D029968:Escherichia coli Proteins; C052985:isochorismic acid; D010088:Oxidoreductases; D044925:Oxidoreductases Acting on CH-CH Group Donors; C061899:entA protein, E coli; D007535:Isomerases; D019751:Intramolecular Transferases; C058486:isochorismate synthase; D002827:Chorismic Acid