Nucleotide sequence of the thrA gene of Escherichia coli.

Abstract

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.

Isolation of the aspartokinase domain of bifunctional aspartokinase I‐homoserine dehydrogenase I from E.coli K12

“…The four subunits are identical [2,3] (Mr 89000); the amino acid polypeptide sequence has been established [4]. Thus, each subunit carries the two catalytic activities which are both subject to feedback inhibition by L-threonine [5,6].…”

“…The complete amino acid sequence of AK I-HDHI being known [4], the exact number of residues corresponding to a given fragment of known molecular mass is easy to calculate. Thus, an N-terminal fragment of M, 24969 would have a sequence corresponding to residues l-235.…”

Looking Back

“…The DNA sequencing methods were not yet available and we had to isolate and sequence tryptic, chymotryptic, and cyanogen bromide peptides from a protein containing 820 amino acid residues. After seven years of hard work, for www.annualreviews.org • Looking Backwhich I shall give here no reference, the methods invented by Maxam and Gilbert and the dideoxynucleotide method of Sanger allowed us to publish the sequence of the threonine-sensitive and of the methioninerepressible aspartokinase-homoserine dehydrogenases (35,56). We gave evidence that the two proteins as well as aspartokinase III, the lysine-sensitive monofunctional protein, derive from a common ancestor (7,35,56).…”

“…After seven years of hard work, for www.annualreviews.org • Looking Backwhich I shall give here no reference, the methods invented by Maxam and Gilbert and the dideoxynucleotide method of Sanger allowed us to publish the sequence of the threonine-sensitive and of the methioninerepressible aspartokinase-homoserine dehydrogenases (35,56). We gave evidence that the two proteins as well as aspartokinase III, the lysine-sensitive monofunctional protein, derive from a common ancestor (7,35,56). The primary sequence of the other two enzymes of the threonine operon, homoserine kinase and threonine synthase, was also determined (24,39).…”

Immunological Characterization of in Vitro Forms of Homoserine Dehydrogenase from Carrot Suspension Cultures

“…A more detailed immunological analysis has shown the distribution of various antigenic determinants between the two AK/ HSDH polypeptides (27). Recently, both of the genes encoding these enzymes have been isolated and sequenced (14,29). Amino acid homology between the HSDH region of the two E. coli proteins is 53%.…”