Characterization and role of the branched-chain aminotransferase (BcaT) isolated from Lactococcus lactis subsp. cremoris NCDO 763.
In Lactococcus lactis, which is widely used as a starter in the cheese industry, the first step of aromatic and branched-chain amino acid degradation is a transamination which is catalyzed by two major aminotransferases. We have previously purified and characterized biochemically and genetically the aromatic aminotransferase, AraT. In the present study, we purified and studied the second enzyme, the branched-chain aminotransferase, BcaT. We cloned and sequenced the corresponding gene and used a mutant, along with the luciferase gene as the reporter, to study the role of the enzyme in amino acid metabolism and to reveal the regulation of gene transcription. BcaT catalyzes transamination of the three branched-chain amino acids and methionine and belongs to class IV of the pyridoxal 5'-phosphate-dependent aminotransferases. In contrast to most of the previously described bacterial BcaTs, which are hexameric, this enzyme is homodimeric. It is responsible for 90% of the total isoleucine and valine aminotransferase activity of the cell and for 50 and 40% of the activity towards leucine and methionine, respectively. The original role of BcaT was probably biosynthetic since expression of its gene was repressed by free amino acids and especially by isoleucine. However, in dairy strains, which are auxotrophic for branched-chain amino acids, BcaT functions only as a catabolic enzyme that initiates the conversion of major aroma precursors. Since this enzyme is still active under cheese-ripening conditions, it certainly plays a major role in cheese flavor development.
Yvon
M
M
Unité de Recherche de Biochimie et Structure des Protéines, I.N.R. A., 78352 Jouy-en-Josas, France. mireille.yvon@diamant.jouy.inra.fr
Chambellon
E
E
Bolotin
A
A
Roudot-Algaron
F
F
D000597:Amino Acids, Branched-Chain / Q000378:metabolism*; D003001:Cloning, Molecular; D003470:Culture Media; D015964:Gene Expression Regulation, Bacterial; D013294:Lactococcus lactis / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016133:Polymerase Chain Reaction; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D000637:Transaminases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D014158:Transcription, Genetic
D000597:Amino Acids, Branched-Chain; D003470:Culture Media; D000637:Transaminases; C019798:branched-chain-amino-acid transaminase