The thrA gene of Escherichia coli codes for a single polypeptide chain having two enzymatic activities required for the biosynthesis of threonine, aspartokinase I and homoserine dehydrogenase I. This gene was cloned in a bacterial plasmid and its complete nucleotide sequence was established. It contains 2460 base pairs that encode for a polypeptide chain of 820 amino acids. The previously determined partial amino acid sequence of this protein is in good agreement with that predicted from the nucleotide sequence. The gene contains an internal sequence that resembles the structure of bacterial ribosome-binding sites, with an AUG preceded by four triplets, each of which can be converted to a nonsense coon by a single mutation. This suggests that the single polypeptide chain was formed by the fusion of two genes and that initiation of translation may occur inside the gene to give a protein fragment having only the homoserine dehydrogenase activity.The thrA gene is the first structural gene of the threonine operon of Escherichia coil K-12 (1, 2). It is composed of two parts, thrAl and thrA2 and codes for a bifunctional enzyme, aspartokinase I-homoserine dehydrbgenase I (EC 2.7.2.4 and EC 1.1.1.3). The native enzyme (3) is a tetramer with each chain carrying, on discrete domains, the aspartokinase I and homoserine dehydrogenase I activities, which are regulated allosterically by L-threonine. Limited proteolysis of the native enzyme leads to a homodimeric fragment having the same COOHterminal sequence as the native enzyme having only the dehydrogenase activity and no longer inhibited by threonine (3). On the other hand, a polypeptide chain synthesized by an ochre mutant that has the same NH2 terminus as the native enzyme assembles as a tetramer having only the aspartokinase activity, still regulated by threonine (3). The determination of the primary structure of aspartokinase I homoserine-dehydrogenase I seemed warranted for a number of reasons. Sequence information was important to understand enzyme structure-function relationships and to elucidate the allosteric properties of the enzyme. It should permit the study of possible evolutionary relationships between the different proteins coded by the threonine operon and the homology with the isofunctional enzymes in E. coli, aspartokinase II-homoserine dehydrogenase II, coded by metL, and aspartokinase III coded by lysC.

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Antibodies raised against the 25-kilodalton (p25) plasmid-encoded polypeptide of Yersinia enterocolitica recognized the homologous protein in the three Yersinia species grown in vitro. This polypeptide was recovered from whole cells as well as from the fluid supernatant of bacteria grown at 37°C in a Ca2+-deficient medium. Furthermore, a 22-kilodalton (p22) plasmid-encoded polypeptide immunologically related to p25 was found only in Y. pestis during early growth. After 30 h of culture, the Y. pestis p25 and p22 were completely degraded, whereas the intensity of the Y. enterocolitica p25 was decreased, but the protein was still detectable in the fluid supernatant. This proteolytic activity was independent of the presence of the virulence plasmid. Some disulfide bonds are probably involved in the quaternary structure of the p25 of the three pathogenic species and of the Y. pestis p22.

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