The primary structure of Escherichia coli K12 aspartokinase I-homoserine dehydrogenase I

Sibilli, Lise; Cossart, Pascale; Chalvignac, M A; Briley, Patricia A.; Costrejean, Jean-Marc; Bras, Gisèle Le; Cohen, Georges N.

doi:10.1016/s0300-9084(78)80712-3

Cited by 6 publications

(3 citation statements)

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“…The purification of the enzyme, the determination of its molecular weight and amino acid composition, the isolation of the cysteine-and tryptophan-containing tryptic peptide, and the purification and sequence of part of the cyanogen bromide fragments have already been described (4)(5)(6)(7)(8)(9).…”

Section: Methodsmentioning

confidence: 99%

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Nucleotide sequence of the thrA gene of Escherichia coli.

Katinka¹,

Cossart²,

Sibilli³

et al. 1980

Proc. Natl. Acad. Sci. U.S.A.

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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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Section: Methodsmentioning

confidence: 99%

“…The determination of the amino acid sequence of the aspartokinase I-homoserine dehydrogenase I was in progress (4)(5)(6)(7)(8)(9) when the chemical and enzymatic DNA sequence determination techniques became available (10,11). It then seemed advantageous to clone the gene and determine its sequence.…”

mentioning

confidence: 99%

Nucleotide sequence of the thrA gene of Escherichia coli.

Katinka¹,

Cossart²,

Sibilli³

et al. 1980

Proc. Natl. Acad. Sci. U.S.A.

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“…it is isolated as a tetramer of molecular weight 4 X 48 000 and corresponds to the N-terminal segment of AK-HDH (Sibilli et al, 1977). The second fragment, the HDH fragment, possesses the dehydrogenase activity and is obtained by limited proteolysis; it is a dimer of molecular weight 2 X 55000 and has the C-terminal amino acid sequence of AK-HDH (Véron et al, 1972).…”

mentioning

confidence: 99%

Independent folding regions in aspartokinase-homoserine dehydrogenase

Dautry‐Varsat¹,

Garel²

1981

Biochemistry

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The folding of two monofunctional fragments of aspartokinase-homoserine dehydrogenase I has been studied. One of these fragments corresponds to the kinase activity and the N-terminal part of the polypeptide chain; the other one corresponds to the dehydrogenase activity and to the C-terminal part of the chain. Both fragments are able to refold into an enzymatically active conformation after complete disruption of their native structure. The kinase fragment folds up into an active monomeric species. The dehydrogenase fragment first folds up into an inactive monomeric species and then associates into an active dimeric species. These two fragments thus correspond to regions capable of autonomous folding. The folding of each of these fragments is compared to that of the corresponding region in the intact aspartokinase--homoserine dehydrogenase I reported previously [Garel, J.R., & Dautry-Varsat, A. (1980) Proc. Natl. Acad. Sci. U.S.A. 77, 3379-3383]. It is concluded that the N-and C-terminal regions of the intact polypeptide chain behave as independent folding units. A model of the sequence of steps involved in the folding process of aspartokinase-homoserine dehydrogenase I is presented; its relevance to the evolution of this protein is also discussed.

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