Characterization of the isocitrate lyase gene from Corynebacterium glutamicum and biochemical analysis of the enzyme.
Isocitrate lyase is a key enzyme in the glyoxylate cycle and is essential as an anaplerotic enzyme for growth on acetate as a carbon source. It is assumed to be of major importance in carbon flux control in the amino acid-producing organism Corynebacterium glutamicum. In crude extracts of C. glutamicum, the specific activities of isocitrate lyase were found to be 0.01 U/mg of protein after growth on glucose and 2.8 U/mg of protein after growth on acetate, indicating tight regulation. The isocitrate lyase gene, aceA, was isolated, subcloned, and characterized. The predicted gene product of aceA consists of 432 amino acids (M(r), 47,228) and shows up to 57% identity to the respective enzymes from other organisms. Downstream of aceA, a gene essential for thiamine biosynthesis was identified. Overexpression of aceA in C. glutamicum resulted in specific activities of 0.1 and 7.4 U/mg of protein in minimal medium containing glucose and acetate, respectively. Inactivation of the chromosomal aceA gene led to an inability to grow on acetate and to the absence of any detectable isocitrate lyase activity. Isocitrate lyase was purified to apparent homogeneity and subjected to biochemical analysis. The native enzyme was shown to be a tetramer of identical subunits, to exhibit an ordered Uni-Bi mechanism of catalysis, and to be effectively inhibited by 3-phosphoglycerate, 6-phosphogluconate, phosphoenolpyruvate, fructose-1,6-bisphosphate, and succinate.
Reinscheid
D J
DJ
Institut für Biotechnologie des Forschungszentrums Jülich, Germany.
Eikmanns
B J
BJ
Sahm
H
H
D000595:Amino Acid Sequence; D001483:Base Sequence; D002413:Cations, Divalent / Q000494:pharmacology; D003352:Corynebacterium / Q000201:enzymology / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics; D015971:Gene Expression Regulation, Enzymologic; D015723:Gene Library; D005798:Genes, Bacterial; D005816:Genetic Complementation Test; D006863:Hydrogen-Ion Concentration; D007522:Isocitrate Lyase / Q000187:drug effects / Q000235:genetics* / Q000378:metabolism; D008670:Metals / Q000494:pharmacology; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D011994:Recombinant Proteins / Q000378:metabolism; D015183:Restriction Mapping; D017421:Sequence Analysis; D017386:Sequence Homology, Amino Acid; D013831:Thiamine / Q000235:genetics
D002413:Cations, Divalent; D008670:Metals; D011994:Recombinant Proteins; D007522:Isocitrate Lyase; D013831:Thiamine