A triglobular model for the polypeptide chain of aspartokinase I-homoserine dehydrogenase I of Escherichia coli

Fazel, Akram M.; Müller, K; G, Le Bras; Garel,; Véron, Michel; Gn, Cohen

doi:10.1021/bi00270a023

Cited by 44 publications

(27 citation statements)

References 24 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The native molecular weight of the normal AKI-HDI protein was 280,000 to 370,000. This was consistent with the hypothesis that it is a tetramer like the E. coli enzyme (10,12). The truncated AKI-HDI molecules containing the N-terminal 462 and 468 amino acid residues were probably dimeric, and the shorter molecules were probably monomeric.…”

Section: Resultssupporting

confidence: 89%

“…The E. coli AKI-HDI enzyme is a large tetrameric protein (10,19). Proteolytic digestion has shown that the AKI and HDI activities are carried by the N-terminal and C-terminal moieties, respectively, and that multimer formation depends on the central region (11,12).Threonine synthesis in Serratia marcescens has been studied extensively (22,23). Both AKI and HDI activities are subject to feedback inhibition by threonine, and expression of the thr operon is multivalently repressed by threonine and isoleucine.…”

mentioning

confidence: 99%

mentioning

confidence: 99%

See 2 more Smart Citations

Nucleotide sequence of the Serratia marcescens threonine operon and analysis of the threonine operon mutations which alter feedback inhibition of both aspartokinase I and homoserine dehydrogenase I

et al. 1993

View full text Add to dashboard Cite

The nucleotide sequence of the Serratia narcescens threonine operon (thrA$,2BC) was determined. Three long open reading frames were identified; these open reading frames code for aspartokinase I (AKI)-homoserine dehydrogenase I (HDI), homoserine kinase, and threonine synthase, in that order. The predicted amino acid sequences of these enzymes were similar to the amino acid sequences of the corresponding enzymes in Escherichia coli. The AKI-HDI protein is apparently a tetramer composed of monomer polypeptides that are 819 amino acids long. A deletion analysis revealed that the central and C-terminal region was responsible for threonine-resistant HDI activity, a monomeric fragment extending from the N terminus to residue 306 was responsible for threonine-resistant AKI activity, and an N-terminal portion containing 468 residues was responsible for threonine-sensitive AKI activity. The thrAlIA21 and thrA15A25 mutations of threonineexcreting strains HNr21 and TLr156, which result in the loss of threonine-mediated feedback inhibition of both AKI activity and HDI activity, cause single amino acid substitutions (Gly to Asp at position 330 and Ser to Phe at position 352, respectively) in the central region of the AKI-HDI protein. The thrA1+A22 mutation of strain HNr59, which results in a threonine-sensitive AKI and a threonine-resistant HDI, also causes a single amino acid substitution (Ala to Thr at position 479).Threonine is synthesized from L-aspartate via five enzymatic reactions. In Escherichia coli, four of the five enzyme activities of this pathway are encoded by the thrAlA2BC operon (39,45). The first and third steps of this pathway are catalyzed by aspartokinase I (AKI) (EC 2.7.2.4) and homoserine dehydrogenase I (HDI) (EC 1.1.1.3); these two enzymes are encoded by the thrA A2 genes in a single polypeptide chain, the AKI-HDI protein. Homoserine kinase (EC 2.7.1.39), the thrB gene product, catalyzes the fourth step. The thrC gene encodes threonine synthase (EC 4.2.99.2), which catalyzes the last step of the pathway. The nucleotide sequences of these thr genes have been reported previously (6,19,35). The E. coli AKI-HDI enzyme is a large tetrameric protein (10,19). Proteolytic digestion has shown that the AKI and HDI activities are carried by the N-terminal and C-terminal moieties, respectively, and that multimer formation depends on the central region (11,12).Threonine synthesis in Serratia marcescens has been studied extensively (22,23). Both AKI and HDI activities are subject to feedback inhibition by threonine, and expression of the thr operon is multivalently repressed by threonine and isoleucine. Therefore, the AKI-HDI protein is a key enzyme of this pathway in S. marcescens.Threonine-producing isolates of S. marcescens Sr4l were obtained previously, and these organisms were found to be insensitive to feedback inhibition for HDI or for both AKI and HDI. Strains HNr21 and HNr59 are resistant to a threonine analog, 0-hydroxynorvaline (23). Both AKI activity and HDI activity in strain HNr21 are resistant to thr...

show abstract

Section: Resultssupporting

confidence: 89%

mentioning

confidence: 99%

See 1 more Smart Citation

Nucleotide sequence of the Serratia marcescens threonine operon and analysis of the threonine operon mutations which alter feedback inhibition of both aspartokinase I and homoserine dehydrogenase I

et al. 1993

View full text Add to dashboard Cite

show abstract

“…y-glutamyl kinase and responsible for the aspartokinase activity (18), the hydrophobic interaction between this region and the central region is probably involved in the allosteric regulation of AKI. A triglobular model for the polypeptide chain of the intact AKI-HDI protein has been proposed (6). These hydrophobic interactions among the AKI, HDI, and central domains may be possible factors in the allosteric regulation of the AKI-HDI protein.…”

mentioning

confidence: 99%

Role of serine 352 in the allosteric response of Serratia marcescens aspartokinase I-homoserine dehydrogenase I analyzed by using site-directed mutagenesis

Omori

Komatsubara

1993

J Bacteriol

View full text Add to dashboard Cite

Aspartokinase I and homoserine dehydrogenase I (AKI-HDI) from Serratia marcescens Sr4l are encoded by the thrA gene as a single polypeptide chain. Previously, a single amino acid substitution of Ser-352 with Phe was shown to produce an AKI-HDI enzyme that is not subject to threonine-mediated feedback inhibition. To determine the role of Ser-352 in the allosteric response, the thrA gene was modified by using site-directed homoserine dehydrogenase II and contains the thrA1SA25 mutation. These strains produce 4 to 24 g of L-threonine per liter in a fermentation medium containing sucrose and urea (10)(11)(12)24). Both AKI and HDI of the strains HNr21 and TLr156 are desensitized for threonine-mediated feedback inhibition (10,24). Strain HNr59 carries threonine-sensitive AKI and threonine-resistant HDI (12).The mutations of these threonine-producing strains have been revealed to result from a single amino acid substitution in the Among these three substitutions, the amino acid replacement of Ser-352 with Phe is the most effective for the desensitization for threonine-mediated feedback inhibition. This paper deals with the changes in the allosteric response of AKI and HDI activities resulting from amino acid substitutions at position 352 in the AKI-HDI protein. MATERUILS AND METHODSBacterial strains and plasmids. The strains and plasmids used are listed in Table 1. E. coli JM109 was used as a host 959 on April 27, 2019 by guest

show abstract

“…On the other hand, limited proteolysis of native AK I/ HDI I under very mild conditions has yielded a variety of proteolytic fragments with principally a homodimeric HDH fragment carrying only the dehydrogenase activity [15,16] and a hybrid fragment composed of the association of a complete subunit with a proteolysed polypeptide [17]. Previous analysis of the kinetics of this proteolytic reaction have led to the hypothesis that dimers of AK I/HDH I were the true substrates of the protease and that the enzyme in its tetrameric state was rather resistant to proteolytic degradation [9, 181. In this paper we describe conditions for the reversible dissociation of AK I/HDH I into dimers and we use their selective sensitivity to limited proteolysis to demonstrate that these dimers reassociate very rapidly into tetramers under the conditions of assay.…”

mentioning

confidence: 99%

Reversible dissociation of aspartokinase I/homoserine dehydrogenase I from Escherichia coli K 12. The active species is the tetramer

et al. 1985

Self Cite

View full text Add to dashboard Cite

Dimers of aspartokinase I/homoserine dehydrogenase 1 from Escherichiu coli K 12 have been isolated under very mild conditions. The dimers which cannot be distinguished from the tetramers by their kinetic properties, reassociate in the presence of potassium ions or L-aspartate. The selective sensitivity of aspartokinase I/homoserine dehydrogenase I to mild proteolytic digestion of dimers has been used to probe the reassociation reaction under the conditions of aspartokinase assay. We demonstrate that rapid reassociation occurs and that the protein species present in the assay when dimers are used to test the activity is tetrameric. These results confirm the previously proposed model for the subunit association of aspartokinase I/homoserine dehydrogenase I.Aspartokinase I/homoserine dehydrogenase I (AK I/ HDH I) is a bifunctional tetrameric protein which catalyses two non-consecutive reactions in the biosynthetic pathway leading from aspartate to threonine and isoleucine [l -31. Each of the four identical polypeptide chains [4, 51 carries the two catalytic activities which are both subject to feedback inhibition by L-threonine [1, 61.Most enzymatic and spectroscopic properties of AK I/ HDH I can be interpreted in terms of a two-state equilibrium between an active and an inactive conformation respectively by different ligands: L-aspartate and K f ions favor the active conformation while L-threonine stabilizes the inactive state of the protein [7, 81. Like many other polymeric enzymes, AK I/HDH I can be found in more than one state of association, and, under certain conditions, the tetramer dissociates into dimers [5]. A number of results indicate that the interprotomeric association involve two types of contacts between subunits and that dissociation of the tetramer into dimers is more easily achieved than dissociation of dimers into monomers [9]. Indeed, dimers retaining at least part of the initial enzymatic activities have been obtained by incubation at low temperature and moderately alkaline pH [5] or in the absence of aspartate and potassium ions [lo, 111. The dissociation is usually accompanied by a loss in sensitivity towards threonine inhibition [12], a result also obtained when the native protein is reacted under mild conditions with sulfhydryl reagents [13]. Dissociation to the monomer is usually accompanied with a loss of enzymatic activity [14].On the other hand, limited proteolysis of native AK I/ HDI I under very mild conditions has yielded a variety of proteolytic fragments with principally a homodimeric HDHCorrespondenc.e to

show abstract

A triglobular model for the polypeptide chain of aspartokinase I-homoserine dehydrogenase I of Escherichia coli

Cited by 44 publications

References 24 publications

Nucleotide sequence of the Serratia marcescens threonine operon and analysis of the threonine operon mutations which alter feedback inhibition of both aspartokinase I and homoserine dehydrogenase I

Nucleotide sequence of the Serratia marcescens threonine operon and analysis of the threonine operon mutations which alter feedback inhibition of both aspartokinase I and homoserine dehydrogenase I

Role of serine 352 in the allosteric response of Serratia marcescens aspartokinase I-homoserine dehydrogenase I analyzed by using site-directed mutagenesis

Reversible dissociation of aspartokinase I/homoserine dehydrogenase I from Escherichia coli K 12. The active species is the tetramer

Contact Info

Product

Resources

About