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 gene from Corynebacterium glutamicum, encoding malate synthase, was isolated, subcloned and expressed in Escherichia coli and C. glutamicum. Sequencing of a 3024 bp DNA fragment containing the aceB gene revealed that it is located close to the isocitrate lyase gene aceA. The two genes 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 82,362. Interestingly, this polypeptide shows only weak identity with malate synthase polypeptides from other organisms and possesses an extra N-terminal sequence of about 170 amino acid residues. Inactivation of the chromosomal aceB gene led to the absence of malate synthase activity and to the inability to grow on acetate, suggesting that only one malate synthase is present in C. glutamicum. The malate synthase was purified from an aceB-overexpressing C. glutamicum strain and biochemically characterized. The native enzyme was shown to be a monomer migrating at an M(r) of about 80,000. By sequencing the N-terminus of malate synthase the predicted translational start site of the enzyme was confirmed. The enzyme displayed Km values of 30 microM and 12 microM for the substrates glyoxylate and acetyl CoA, respectively. Oxalate, glycolate and ATP were found to be inhibitors of malate synthase activity. The present study provides evidence that the malate synthase from C. glutamicum is functionally similar to other malate synthase enzymes but is different both in size and primary structure.
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; D003001:Cloning, Molecular; D003352:Corynebacterium / Q000201:enzymology / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics; D005798:Genes, Bacterial / Q000235:genetics*; D007522:Isocitrate Lyase / Q000235:genetics; D008292:Malate Synthase / Q000235:genetics* / Q000302:isolation & purification; D008969:Molecular Sequence Data; D016296:Mutagenesis; D011994:Recombinant Proteins / Q000302:isolation & purification; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid
D011994:Recombinant Proteins; D008292:Malate Synthase; D007522:Isocitrate Lyase