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 essent ial as an anaplerotic enzyme for growth on acetate asa carbon source. It is assumed to be of major importance in carbon Bur control in the a mino acid-producing organism Corynebacterium glutamicum. In crude extr acts of C. glutamicum, the specific activities of isocitrate lyase wer e found to be 0.01 U/mg of protein after growth on glucose and 2.8 U/m g of protein after growth on acetate, indicating tight regulation. The isocitrate lyase gene, aceA, was isolated, subcloned, and characteriz ed. 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 fro m other organisms. Downstream of aceA, a gene essential for thiamine b iosynthesis was identified. Overexpression of aceA in C. glutamicum re sulted in specific activities of 0.1 and 7.4 U/mg of protein in minima l 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 biochemic al 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, p hosphoenolpyruvate, fructose-1,6-bisphosphate, and succinate.