CLONING, SEQUENCE-ANALYSIS, EXPRESSION, AND INACTIVATION OF THE CORYNEBACTERIUM-GLUTAMICUM-ICD GENE ENCODING ISOCITRATE DEHYDROGENASE AND BIOCHEMICAL-CHARACTERIZATION OF THE ENZYME

NADP(+)-dependent isocitrate dehydrogenase (ICD) is an important enzym e of the intermediary metabolism, as it controls the carbon flux withi n the citric acid cycle and supplies the cell,vith 2-oxoglutarate and NADPH for biosynthetic purposes. In the amino acid-producing organism Corynebacterium glutamicum, the specific activity of ICD was independe nt of the growth substrate and of the growth phase at approximately 1 U/mg, indicating that this enzyme is constitutively formed. The ICD ge ne, icd, was isolated, subcloned on a plasmid, and introduced into C. glutamicum. Compared with the wild type, the recombinant strains showe d up to 10-fold-higher specific ICD activities. The nucleotide sequenc e of a 3,595-bp DNA fragment containing the icd gene was determined. T he predicted gene product of icd consists of 739 amino acids (M(r) = 8 0,091) and showed 58.5% identity with the monomeric ICD isozyme II fro m Vibrio sp. strain ABE-1 but no similarity to any known ICD of the di meric type. Inactivation of the chromosomal icd gene led to glutamate auxotrophy and to the absence of any detectable ICD activity, suggesti ng that only a single ICD is present in C. glutamicum. From an icd-ove rexpressing C. glutamicum strain, ICD was purified and biochemically c haracterized. The native ICD was found to be a monomer; to be specific for NADP(+); to be weakly inhibited by oxaloacetate, 2-oxoglutarate, and citrate; and to be severely inhibited by oxaloacetate plus glyoxyl ate. The data indicate that ICD from C. glutamicum is structurally sim ilar to ICDs from bacteria of the genera Vibrio, Rhodomicrobium and Az otobacter but different from all other known procaryotic and eucaryoti c ICDs.