Yvon M, Chambellon E, Bolotin A, Roudot-Algaron F
Unité de Recherche de Biochimie et Structure des Protéines, I.N.R. A., 78352 Jouy-en-Josas, France.
Appl Environ Microbiol. 2000 Feb;66(2):571-7. doi: 10.1128/AEM.66.2.571-577.2000.
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.
乳酸乳球菌被广泛用作奶酪工业中的发酵剂，芳香族和支链氨基酸降解的第一步是转氨作用，由两种主要的转氨酶催化。我们之前已经对芳香族转氨酶AraT进行了生化和遗传纯化及表征。在本研究中，我们纯化并研究了第二种酶，即支链转氨酶BcaT。我们克隆并测序了相应基因，并使用一个突变体以及荧光素酶基因作为报告基因，来研究该酶在氨基酸代谢中的作用，并揭示基因转录的调控机制。BcaT催化三种支链氨基酸和蛋氨酸的转氨作用，属于磷酸吡哆醛依赖性转氨酶的IV类。与大多数先前描述的细菌BcaT（为六聚体）不同，这种酶是同型二聚体。它负责细胞中90%的异亮氨酸和缬氨酸转氨酶总活性，以及分别50%和40%的亮氨酸和蛋氨酸转氨酶活性。BcaT最初的作用可能是生物合成性的，因为其基因的表达受到游离氨基酸尤其是异亮氨酸的抑制。然而，在对支链氨基酸营养缺陷的乳制品菌株中，BcaT仅作为一种分解代谢酶，启动主要风味前体的转化。由于这种酶在奶酪成熟条件下仍然具有活性，它肯定在奶酪风味形成中起主要作用。
