Metabolic engineering of a non-allosteric citrate synthase in an Escherichia coli citrate synthase mutant.
This study examined the organization of the Krebs tricarboxylic acid (TCA) cycle by metabolic engineering and high-resolution 13C NMR. The oxidation of [1,2,3-13C]propionate to glutamate via the TCA cycle was measured in wild-type (WT) and a citrate synthase mutant (CS-) strain of Escherichia coli transformed with allosteric E. coli citrate synthase (ECCS) or non-allosteric pig citrate synthase (PCS). The 13C fractional enrichment in glutamate C-2, C-3, and C-4 in ECCS and PCS were similar; although quantitative differences in total citrate synthase activity and total C-4 labeling of glutamate were observed in ECCS and PCS. Allosteric ECCS cells contained 10-fold less total enzyme activity than PCS but only 50% less total labeling in glutamate C-4 and equivalent doubling times. The observed spectra were mathematically fitted using an iterative procedure (TCACALC) and yielded an acetate/succinyl-CoA flux ratio of 10 for both ECCS and PCS, a result that is in agreement with the isotopomer analyses of the 13C spectra of cells presented with [3-13C]propionate or [2-13C]propionate. The results are consistent with the presence of an allosteric citrate synthase in ECCS and a non-allosteric citrate synthase in PCS. The former maintains TCA cycle flux via alternative propionate pathways activated by positive allosteric mechanisms and the latter via elevated enzyme levels.
Evans
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Department of Veterans Affairs Medical Center, Southwestern Medical Center, Dallas 75216, USA.
D000494:Allosteric Regulation; D000495:Allosteric Site; D000818:Animals; D001426:Bacterial Proteins / Q000378:metabolism*; D002950:Citrate (si)-Synthase / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development; D009682:Magnetic Resonance Spectroscopy; D009154:Mutation; D010084:Oxidation-Reduction; D011422:Propionates / Q000378:metabolism*; D013552:Swine
D001426:Bacterial Proteins; D011422:Propionates; D002950:Citrate (si)-Synthase