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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.

The enzymatic degradation of amino acids in cheese is believed to generate aroma compounds and therefore to be involved in the complex process of cheese flavor development. In lactococci, transamination is the first step in the degradation of aromatic and branched-chain amino acids which are precursors of aroma compounds. Here, the major aromatic amino acid aminotransferase of a Lactococcus lactis subsp. cremoris strain was purified and characterized. The enzyme transaminates the aromatic amino acids, leucine, and methionine. It uses the ketoacids corresponding to these amino acids and alpha-ketoglutarate as amino group acceptors. In contrast to most bacterial aromatic aminotransferases, it does not act on aspartate and does not use oxaloacetate as second substrate. It is essential for the transformation of aromatic amino acids to flavor compounds. It is a pyridoxal 5'-phosphate-dependent enzyme and is composed of two identical subunits of 43.5 kDa. The activity of the enzyme is optimal between pH 6.5 and 8 and between 35 and 45 degrees C, but it is still active under cheese-ripening conditions.

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Bacillus subtilis can use methionine as the sole sulfur source, indicating an efficient conversion of methionine to cysteine. To characterize this pathway, the enzymatic activities of CysK, YrhA and YrhB purified in Escherichia coli were tested. Both CysK and YrhA have an O-acetylserine-thiol-lyase activity, but YrhA was 75-fold less active than CysK. An atypical cystathionine ␤-synthase activity using O-acetylserine and homocysteine as substrates was observed for YrhA but not for CysK. The YrhB protein had both cystathionine lyase and homocysteine ␥-lyase activities in vitro. Due to their activity, we propose that YrhA and YrhB should be renamed MccA and MccB for methionine-to-cysteine conversion. Mutants inactivated for cysK or yrhB grew similarly to the wild-type strain in the presence of methionine. In contrast, the growth of an ⌬yrhA mutant or a luxS mutant, inactivated for the S-ribosyl-homocysteinase step of the S-adenosylmethionine recycling pathway, was strongly reduced with methionine, whereas a ⌬yrhA ⌬cysK or cysE mutant did not grow at all under the same conditions. The yrhB and yrhA genes form an operon together with yrrT, mtnN, and yrhC. The expression of the yrrT operon was repressed in the presence of sulfate or cysteine. Both purified CysK and CymR, the global repressor of cysteine metabolism, were required to observe the formation of a protein-DNA complex with the yrrT promoter region in gel-shift experiments. The addition of O-acetyl-serine prevented the formation of this protein-DNA complex.Methionine plays a central role in a variety of cellular functions. This amino acid is the universal initiator of protein synthesis, and its derivative, S-adenosylmethionine (AdoMet), is involved in several cellular processes, including methylation and polyamine biosynthesis. Methionine synthesis and degradation are, therefore, tightly regulated.Bacillus subtilis, like several microorganisms (21, 28, 55), can use sulfate for the synthesis of organic sulfur metabolites, mostly cysteine, methionine, and AdoMet. As in enterobacteria (28), the sulfate assimilation pathway of B. subtilis involves uptake and activation of inorganic sulfate, followed by stepwise reduction to sulfide (Fig.