MALATE SYNTHASE FROM CORYNEBACTERIUM-GLUTAMICUM - SEQUENCE-ANALYSIS OF THE GENE AND BIOCHEMICAL-CHARACTERIZATION OF THE ENZYME

Malate synthase is one of the key enzymes of the glyoxylate cycle and is essential for growth on acetate as sole carbon source. The aceB gen e from Corynebacterium glutamicum, encoding malate synthase, was isola ted, subcloned and expressed in Escherichia coli and C. glutamicum. Se quencing of a 3024 bp DNA fragment containing the aceB gene revealed t hat it is located close to the isocitrate lyase gene aceA. The two gen es are separated by 597 bp and are transcribed in divergent directions . The predicted aceB gene product consists of 739 amino acids with an M(r) of 82362. Interestingly. this polypeptide shows only weak identit y with malate synthase polypeptides from other organisms and possesses an extra N-terminal sequence of about 170 amino acid residues. Inacti vation of the chromosomal aceB gene led to the absence of malate synth ase activity and to the inability to grow on acetate, suggesting that only one malate synthase is present in C. glutamicum. The malate synth ase was purified from an aceB-overexpressing C. glutamicum strain and biochemically characterized. The native enzyme was shown to be a monom er migrating at an M(r) of about 80000. By sequencing the N-terminus o f malate synthase the predicted translational start site of the enzyme was confirmed. The enzyme displayed K-m values of 30 mu M and 12 mu M for the substrates glyoxylate and acetyl CoA, respectively. Oxalate. glycolate and ATP were found to be inhibitors of malate synthase activ ity. The present study provides evidence that the malate synthase from C. glutamicum is functionally similar to other malate synthase enzyme s but is different both in size and primary structure.