Regulation by methionine of the synthesis of third aspartokinase and of a second homoserine dehydrohenase in Escherichia coli K 12

Patte, Jean‐Claude; Bras, Gisèle Le; Cohen, Georges N.

doi:10.1016/0304-4165(67)90069-4

Cited by 127 publications

(53 citation statements)

References 25 publications

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“…A single organism can produce one, two, or even three different aspartokinases. For example, E. coli has three genes encoding three different aspartokinases (50), and an aspartokinase null mutant of E. coli must have mutations in all three genes (68). Likewise, Bacillus subtilis has three aspartokinase isoenzymes, all of which appear to be produced from different genes (71).…”

Section: Discussionmentioning

confidence: 99%

Biosynthesis of diaminopimelate, the precursor of lysine and a component of peptidoglycan, is an essential function of Mycobacterium smegmatis

1996

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Diaminopimelate (DAP) is a unique metabolite used for both the biosynthesis of lysine in bacteria and the construction of the peptidoglycan of many species of bacteria, including mycobacteria. DAP is synthesized by bacteria as part of the aspartate amino acid family, which includes methionine, threonine, isoleucine, and lysine. Aspartokinase, the first enzyme in this pathway, is encoded by the ask gene in mycobacteria. Previous attempts to disrupt this gene in Mycobacterium smegmatis were unsuccessful, even when the cells were supplied with all the members of the aspartate family, suggesting that unlike other bacteria, mycobacteria may have an absolute requirement for this pathway even when growing in rich medium containing DAP. The purpose of this study was to determine if the ask gene and the aspartate pathway are essential to M. smegmatis. This study describes a test for gene essentiality in mycobacteria, utilizing a counterselectable marker (streptomycin resistance) in conjunction with a specially constructed merodiploid strain. We have used this system to show that the ask gene could not be disrupted in wild-type M. smegmatis, using selective rich medium supplemented with DAP unless there was an extra copy of ask provided elsewhere in the chromosome. Disruption of ask was also possible in a lysine auxotroph incapable of converting DAP to lysine. The ask mutant, mc 2 1278 (ask1::aph), exhibits multiple auxotrophy (Met ؊ , Thr, and Lys ؊ ) and is complemented by the ask gene. This is the first description of DAP auxotrophy in mycobacteria. The ask mutant lyses when deprived of DAP in culture, a characteristic which can be exploited for the reproducible preparation of protoplasts and mycobacterial extracts. The evidence presented here indicates that the aspartate pathway is essential to M. smegmatis and that DAP is the essential product of this pathway.Globally, tuberculosis is the leading cause of death in adults due to an infectious organism (20). It is estimated that 90 million new tuberculosis cases resulting in 30 million deaths can be expected during the last decade of this century (51). The resurgence of tuberculosis in developing nations (62), the appearance of multidrug-resistant strains of Mycobacterium tuberculosis (62), and the problem of tuberculosis in the immunocompromised (28) call for further study of mycobacteria. More knowledge about the basic biology of mycobacteria is needed in order to develop a deeper understanding of the pathogenesis of mycobacterial diseases. Furthermore, identification of biological processes specifically essential for the growth and development of mycobacteria will allow the rational design of drugs to inhibit those processes.The complex cell envelope of the mycobacteria is an outstanding feature of these organisms (13). The envelope is composed of a variety of complex lipids including the long-chain mycolic acids and unique polysaccharides such as arabinogalactan and arabinomannan (7). These components contribute to the hydrophobic nature of the mycobacterial cell s...

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Section: Discussionmentioning

confidence: 99%

Biosynthesis of diaminopimelate, the precursor of lysine and a component of peptidoglycan, is an essential function of Mycobacterium smegmatis

1996

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“…Patte et al [10] reported 1 mM and 2 mM respectively, but with a different enzymic assay. For the equilibrium constant, the fitting of the time course of the aspp concentrations (Figure 2), taking into account the hydrolysis of aspp, gives a value of 6.4i10 −% (see the Experimental section), with a rate constant for the hydrolysis of 0.049 min −" .…”

Section: Ak Imentioning

confidence: 99%

“…AK I, a bifunctional enzyme incorporating homoserine dehydrogenase (HDH) [9], has had its substrate kinetics (aspartate and ATP) studied at pH 8.0 and 27 mC by Patte et al [9,10] and at the same pH and 30 mC by Angeles and Viola [11]. This was one of the few studies where a product-inhibition parameter (the K i for ADP) was measured, and is significant in this case because the equilibrium constant of the reaction (3.5i10 −% at pH 8.0 and 15 mC [12]) favours the formation of aspartate and ATP.…”

Section: Figure 1 Threonine Pathway From Aspartate In E Colimentioning

confidence: 99%

An integrated study of threonine-pathway enzyme kinetics in Escherichia coli

Chassagnole

Raïs

QUENTIN³

et al. 2001

Biochemical Journal

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We have determined the kinetic parameters of the individual steps of the threonine pathway from aspartate in Escherichia coli under a single set of experimental conditions chosen to be physiologically relevant. Our aim was to summarize the kinetic behaviour of each enzyme in a single tractable equation that takes into account the effect of the products as competitive inhibitors of the substrates in the forward reaction and also, when appropriate (e.g. near-equilibrium reactions), as substrates of the reverse reactions. Co-operative feedback inhibition by threonine and lysine was also included as necessary. We derived the simplest rate equations that describe the salient features of the enzymes in the physiological range of metabolite concentrations in order to incorporate them ultimately into a complete model of the threonine pathway, able to predict quantitatively the behaviour of the pathway under natural or engineered conditions.

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“…With my students Jean-Claude Patte, Françoise Falcoz-Kelly, Paolo Truffa-Bachi, and Joël Janin, we demonstrated that one of the two aspartate kinases, uncovered by Stadtman and myself, and homoserine dehydrogenase were carried by a single bifunctional protein, aspartokinase I-homoserine dehydrogenase I (17,41). Jean-Claude Patte found a third aspartokinase that was also a bifunctional protein, aspartokinase II-homoserine dehydrogenase II, the synthesis of which was under the control of methionine (40). Owing to the skepticism of some of our colleagues, we decided to become protein chemists to prove our point.…”

Section: Gif Sur Yvette (1960-1969): Multifunctional Proteinsmentioning

confidence: 69%

Looking Back

Cohen

2005

Annu. Rev. Microbiol.

Self Cite

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My encounter with Jacques Monod has shaped my scientific career. After a short incursion in the biochemistry of strict anaerobes, and after elucidating the biosynthetic pathway leading from aspartate to threonine in Escherichia coli, I joined his laboratory. With him and Howard Rickenberg, I discovered the stereospecific permeability of galactosides and amino acids (permeases). After this intermezzo, I returned to the analysis of biosynthetic pathways and of their regulation by allosteric feedback inhibition and repression in E. coli. Among others, my studies led to the discovery of the tryptophan and methionine repressors, to the incorporation of amino acid analogues in proteins, including selenomethionine (which much later led to progress in protein crystallography), to the definition of isofunctional and multifunctional enzymes, and to the elucidation of the primary structure of most of the enzymes leading to threonine and methionine.

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Regulation by methionine of the synthesis of third aspartokinase and of a second homoserine dehydrohenase in Escherichia coli K 12

Cited by 127 publications

References 25 publications

Biosynthesis of diaminopimelate, the precursor of lysine and a component of peptidoglycan, is an essential function of Mycobacterium smegmatis

Biosynthesis of diaminopimelate, the precursor of lysine and a component of peptidoglycan, is an essential function of Mycobacterium smegmatis

An integrated study of threonine-pathway enzyme kinetics in Escherichia coli

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