Influence of threonine exporters on threonine production in Escherichia coli

Threonine production in Escherichia coli threonine producer strains is enhanced by overexpression of the E. coli rhtB and rhtC genes or by heterologous overexpression of the gene encoding the Corynebacterium glutamicum threonine excretion carrier, thrE. Both E. coli genes give rise to a threonine-resistant phenotype when overexpressed, and they decrease the accumulation of radioactive metabolites derived from [(14)C] L-threonine. The evidence presented supports the conclusion that both RhtB and RhtC catalyze efflux of L-threonine and other structurally related neutral amino acids, but that the specificities of these two carriers differ substantially.     

Amination in E. coli strains effectively producing threonine

The effect of the mutation of threonine and homoserine resistance (thrr) on the activity of the enzymes catalysing the biosynthesis of glutamic acid, glutamate synthase (EC 1.4.1.13) and glutamate dehydrogenase (EC 1.4.1.4), and on the productivity of a threonine-producing E. coli strain obtained by gene engineering was being studied. The resistance to threonine was found to correlate well with the increasing activities of the abovementioned enzymes and with a higher productivity of the E. coli strain.     

Cloning and nucleotide sequences of the homoserine dehydrogenase genes (hom) and the threonine synthase genes (thrC) of the gram-negative obligate methylotroph Methylobacillus glycogenes.

We have cloned the homoserine dehydrogenase genes (hom) from the gram-negative obligate methylotrophs Methylobacillus glycogenes ATCC 21276 and ATCC 21371 by complementation of an Escherichia coli homoserine dehydrogenase-deficient mutant. The 4.15-kb DNA fragment cloned from M. glycogenes ATCC 21371 also complemented an E. coli threonine synthase-deficient mutant, suggesting the DNA fragment contained the thrC gene in addition to the hom gene. The homoserine dehydrogenases expressed in the E. coli recombinants were hardly inhibited by L-threonine, L-phenylalanine, or L-methionine. However, they became sensitive to the amino acids after storage at 4 degrees C for 4 days as in M. glycogenes. The structures of the homoserine dehydrogenases overexpressed in E. coli were thought to be different from those in M. glycogenes, probably in subunit numbers of the enzyme, and were thought to have converted to the correct structures during the storage. The nucleotide sequences of the hom and thrC genes were determined. The hom genes of M. glycogenes ATCC 21276 and ATCC 21371 encode peptides with M(r)s of 48,225 and 44,815, respectively. The thrC genes were located 50 bp downstream of the hom genes. The thrC gene of ATCC 21371 encodes a peptide with an M(r) of 52,111, and the gene product of ATCC 21276 was truncated. Northern (RNA) blot analysis suggests that the hom and thrC genes are organized in an operon. Significant homology between the predicted amino acid sequences of the hom and thrC genes and those from other microorganisms was found.     

Target of serine inhibition in Escherichia coli

L-serine has long been known to inhibit growth of Escherichia coli cells cultured in minimal medium supplemented with glucose, lactate, or another carbohydrate as the sole source of carbon. However, the target of serine inhibition was not known. The growth inhibition was released by adding isoleucine, 2-ketobutyric acid, threonine or homoserine, but not by aspartate. Thus the inhibition site must be between aspartate and homoserine in the isoleucine biosynthetic pathway. We found that homoserine dehydrogenase I was strongly inhibited by serine. We isolated serine-resistant mutants, and found that in these mutants homoserine dehydrogenase I was resistant to serine. Thus, we conclude that the target of serine inhibition in Escherichia coli is homoserine dehydrogenase I.     

Effects of deregulation of methionine biosynthesis on methionine excretion in Escherichia coli

Several regulators of methionine biosynthesis have been reported in Escherichia coli, which might represent barriers to the production of excess l-methionine (Met). In order to examine the effects of these factors on Met biosynthesis and metabolism, deletion mutations of the methionine repressor (metJ) and threonine biosynthetic (thrBC) genes were introduced into the W3110 wild-type strain of E. coli. Mutations of the metK gene encoding S-adenosylmethionine synthetase, which is involved in Met metabolism, were detected in 12 norleucine-resistant mutants. Three of the mutations in the metK structural gene were then introduced into metJ and thrBC double-mutant strains; one of the resultant strains was found to accumulate 0.13 g/liter Met. Mutations of the metA gene encoding homoserine succinyltransferase were detected in alpha-methylmethionine-resistant mutants, and these mutations were found to encode feedback-resistant enzymes in a 14C-labeled homoserine assay. Three metA mutations were introduced, using expression plasmids, into an E. coli strain that was shown to accumulate 0.24 g/liter Met. Combining mutations that affect the deregulation of Met biosynthesis and metabolism is therefore an effective approach for the production of Met-excreting strains.     

Assessing the roles of essential functional groups in the mechanism of homoserine succinyltransferase

Homoserine acyltransferases catalyze the commitment step to methionine and other important biological precursors which make this class of enzymes essential for the survival of bacteria, plants and fungi. This class of enzymes is not found in humans, making them an attractive new target for antimicrobial design. Homoserine O-succinyltransferase (HST) is a representative from this class, with little known about the key amino acids involved in substrate specificity and catalysis. HST from Escherichia coli has been cloned, purified and kinetically characterized. Through site-directed mutagenesis and steady-state kinetic studies the residues that comprise a catalytic triad for HST, the catalytic cysteine nucleophile, an active site acid-base histidine, and the base orienting glutamate, have been identified and characterized. Several residues which confer substrate specificity for both homoserine and succinyl-CoA have also been identified and kinetically evaluated. Mutations of an active site glutamate to either aspartate or alanine drastically increase the K(m) for homoserine, assigning this glutamate to a binding role for the alpha-amino group of homoserine. An active site arginine orients the carboxyl moiety of homoserine, while the carboxyl moiety of succinyl-CoA is positioned for catalysis by a lysine residue. Removing functionality at either of these positions alters the enzyme's ability to effectively utilize homoserine or succinyl-CoA, respectively, reflected in an increased K(m) and decreased catalytic efficiency. The data presented here provides new details of the catalytic mechanism of succinyltransferases, resolves a controversy between alternative mechanistic hypotheses, and provides a starting point for the development of selective inhibitors of HST.     

Immunological cross reactivity of four enzymes involved in the biosynthetic pathway of lysine, methionine and threonine in Escherichia coli K12.

In Escherichia coli K12 the biosynthetic pathway of lysine, methionine and threonine is characterized by three isofunctional aspartokinases and two homoserine dehydrogenases. A single polypeptide chain carries the threonine-sensitive aspartokinase and homoserine dehydrogenase (AK I-HDH I), and a different polypeptide chain carries the methionine-repressible aspartokinase and homoserine dehydrogenase (AK II-HDH II). Immuno-adsorbants prepared with rabbit antibodies against AK I-HDH I bind the lysine-sensitive aspartokinase (AK III), the AK II-HDH II, and the homoserine kinase (HSK), an enzyme of the threonine biosynthetic pathway. Saturation of the immunoadsorbant with AK I-HDH I results in a decreased binding capacity for the other enzymes. Displacement of bound AK III or HSK can be obtained with pure AK I-HDH I, showing that the affinity of the antibodies to homologous antigens is higher than to heterologous ones. Immunoadsorbants prepared with anti-HSK antibodies show the same type of recognition: binding of the three aspartkinases and a capacity to displace the heterologous antigens bound. Accordingly, the same antibodies, implicated in the binding of the homologous antigen, bind the other enzymes. None of the other enzymes of the pathway, or the other kinases tested are recognized by the two immunoadsorbants. It can be postulated that in E. coli K12, duplication of a common ancestor gene gave rise to the three aspartokinases and to the homoserine kinase; two of the genes coding for the aspartokinases fused with those coding for the homoserine dehydrogenases. Indicating that only few epitopes are shared by these enzymes, by conventional immuno-diffusion techniques no precipitation lines appeared with antibodies against AK I-HDH I and the other proteins.     

Expression of the metA gene of Escherichia coli K-12 in recombinant plasmids

Abstract The expression of the metA gene coding for the first enzyme in the methionine biosynthethic pathway was studied in wild-type and in deregulated strains of Escherichia coli K-12 carrying the gene on multicopy plasmids.
We looked at (a) in vitro activity of the metA product—The enzyme homoserine transsuccinylase (HTS); (b) resistance of cells carrying metA plasmids to the analogue α-methylmethionine which specifically inhibits HTS, and (c) the metA polypeptide in mini cells.
The results indicate that the M _r value of the polypeptide synthesized by the metA gene is 40 000. The synthesis of HTS, even when the metA gene is cloned on a multicopy plasmid, is under the negative control of the regulatory metJ gene.     

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

Dimers of aspartokinase I/homoserine dehydrogenase I from Escherichia 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.     

The methionine biosynthetic pathway from homoserine in Pseudomonas putida involves the metW, metX, metZ, metH and metE gene products

Biosynthesis of methionine from homoserine in Pseudomonas putida takes place in three steps. The first step is the acylation of homoserine to yield an acyl-L-homoserine. This reaction is catalyzed by the products of the metXW genes and is equivalent to the first step in enterobacteria, gram-positive bacteria and fungi, except that in these microorganisms the reaction is catalyzed by a single polypeptide (the product of the metA gene in Escherichia coli and the met5 gene product in Neurospora crassa). In Pseudomonas putida, as in gram-positive bacteria and certain fungi, the second and third steps are a direct sulfhydrylation that converts the O-acyl-L-homoserine into homocysteine and further methylation to yield methionine. The latter reaction can be mediated by either of the two methionine synthetases present in the cells.     

Cloning and structural-functional analysis in Escherichia coli of genes of glutamate-producing corynebacteria controlling biosynthesis of amino acids of aspartic acid series

A library of EcoRI DNA fragments from Brevibacterium flavum was constructed using plasmid vector. The genes complementing ThrA2 and ThrB mutations in Escherichia coli were identified in the library. The gene thrA2 of B. flavum codes for mutant enzyme homoserine dehydrogenase insensitive to inhibition by threonine. The genes thrA2 and thrB are localized wihtin the EcoRI fragment 4.1 kb long and are expressed under the control of their own promoters in E. coli cells. Structural and functional analysis of cloned C. glutamicum gene ilvA was performed. The gene of C. glutamicum complemented ilvA mutation in E. coli and appeared to be localized within the EcoRI--SacI DNA fragment 1.6 kb in size. Using E. coli minicells we have demonstrated that the gene ilvA of C. glutamicum controls the synthesis of polypeptide of relative molecular mass 50 kD.     

Identification of a copper-responsive two-component system on the chromosome of Escherichia coli K-12

Using a genetic screen we have identified two chromosomal genes, cusRS (ylcA ybcZ), from Escherichia coli K-12 that encode a two-component, signal transduction system that is responsive to copper ions. This regulatory system is required for copper-induced expression of pcoE, a plasmid-borne gene from the E. coli copper resistance operon pco. The closest homologs of CusR and CusS are plasmid-borne two-component systems that are also involved in metal responsive gene regulation: PcoR and PcoS from the pco operon of E. coli; CopR and CopS from the cop operon, which provides copper resistance to Pseudomonas syringae; and SilR and SilS from the sil locus, which provides silver ion resistance to Salmonella enterica serovar Typhimurium. The genes cusRS are also required for the copper-dependent expression of at least one chromosomal gene, designated cusC (ylcB), which is allelic to the recently identified virulence gene ibeB in E. coli K1. The cus locus may comprise a copper ion efflux system, because the expression of cusC is induced by high concentrations of copper ions. Furthermore, the translation products of cusC and additional downstream genes are homologous to known metal ion antiporters.     

A single amino acid change is responsible for evolution of acyltransferase specificity in bacterial methionine biosynthesis

Bacteria and yeast rely on either homoserine transsuccinylase (HTS, metA) or homoserine transacetylase (HTA; met2) for the biosynthesis of methionine. Although HTS and HTA catalyze similar chemical reactions, these proteins are typically unrelated in both sequence and three-dimensional structure. Here we present the 2.0 A resolution x-ray crystal structure of the Bacillus cereus metA protein in complex with homoserine, which provides the first view of a ligand bound to either HTA or HTS. Surprisingly, functional analysis of the B. cereus metA protein shows that it does not use succinyl-CoA as a substrate. Instead, the protein catalyzes the transacetylation of homoserine using acetyl-CoA. Therefore, the B. cereus metA protein functions as an HTA despite greater than 50% sequence identity with bona fide HTS proteins. This result emphasizes the need for functional confirmation of annotations of enzyme function based on either sequence or structural comparisons. Kinetic analysis of site-directed mutants reveals that the B. cereus metA protein and the E. coli HTS share a common catalytic mechanism. Structural and functional examination of the B. cereus metA protein reveals that a single amino acid in the active site determines acetyl-CoA (Glu-111) versus succinyl-CoA (Gly-111) specificity in the metA-like of acyltransferases. Switching of this residue provides a mechanism for evolving substrate specificity in bacterial methionine biosynthesis. Within this enzyme family, HTS and HTA activity likely arises from divergent evolution in a common structural scaffold with conserved catalytic machinery and homoserine binding sites.     

Development of the vector-host system in Corynebacterium. Cloning and expression of homoserine dehydrogenase and homoserine kinase genes in Corynebacterium cells

Novel cloning vectors for glutamic acid producing bacteria have been constructed. The cryptic plasmid pBO1 (4.4 kb) from Brevibacterium sp. recombined with the plasmid pACYC184 (4.0 kb) from Escherichia coli was used to produce composite plasmid named pKA1. The plasmid could propagate and express the Cm-r phenotype in E. coli and coryneform glutamic acid producing bacteria Br. flavum, C. glutamicum, Br. lactofermentum. The pKA1 plasmid and its variants deleted within non-essential plasmid regions with unique restriction sites HindIII, SalGI, SphI were used in cloning experiments. The genes coding for threonine biosynthesis of C. glutamicum and Br. flavum were subcloned into shuttle vectors in C. glutamicum cells. Recombinant plasmids were introduced into protoplasts by polyethylenglycol-mediated transformation of plasmid DNAs. It was shown that the presence of plasmids containing the Br. flavum thrA2 gene in C. glutamicum (thrB) caused 10-fold increase in homoserine dehydrogenase activity, as compared to that of wild type strain, and in homoserine production.     

Nucleotide sequence of the thrB gene of E. coli, and its two adjacent regions; the thrAB and thrBC junctions.

We have sequenced a DNA fragment containing the Escherichia coli thrA-thrB junction, the complete thrB gene and the thrB-thrC junction. The intergenic sequence thrA and thrB is only one base pair. The coding region for homoserine kinase is 927 base pairs long. It is followed by 114 base pair segment in an open reading frame predicting that thrC begins just after non-sense codon of thrB. The presence at the end of thrA and of thrB of sequences that can pair with the 3' end of the 16 S ribosomal RNA suggests that reinitiation of translation occurs at the end of the two genes. The deduced aminoacid sequence for homoserine kinase shows no striking homology with aspartokinase I homoserine dehydrogenase I.     

Structure of the Escherichia coli quorum sensing protein SdiA: activation of the folding switch by acyl homoserine lactones

The three-dimensional structure of a complex between the N-terminal domain of the quorum sensing protein SdiA of Escherichia coli and a candidate autoinducer N-octanoyl-L-homoserine lactone (C8-HSL) has been calculated in solution from NMR data. The SdiA-HSL system shows the "folding switch" behavior that has been seen for quorum-sensing factors produced by other bacterial species. In the presence of C8-HSL, a significant proportion of the SdiA protein is produced in a folded, soluble form in an E.coli expression system, whereas in the absence of acyl homoserine lactones, the protein is expressed into insoluble inclusion bodies. In the three-dimensional structure, the autoinducer molecule is sequestered in a deep pocket in the hydrophobic core, forming an integral part of the core packing of the folded SdiA. The NMR spectra of the complex show that the bound C8-HSL is conformationally heterogeneous, either due to motion within the pocket or to heterogeneity of the bound structure. The C8-HSL conformation is defined by NOEs to the protein only at the terminal methyl group of the octanoyl chain. Unlike other well-studied bacterial quorum sensing systems such as LuxR of Vibrio fischeri and TraR of Agrobacterium tumefaciens, there is no endogenous autoinducer for SdiA in E.coli: the E.coli genome does not contain a gene analogous to the LuxI and TraI autoinducer synthetases. We show that two other homoserine lactone derivatives are also capable of acting as a folding-switch autoinducers for SdiA. The observed structural heterogeneity of the bound C8-HSL in the complex, together with the variety of autoinducer-type molecules that can apparently act as folding switches in this system, are consistent with the postulated biological function of the SdiA protein as a detector of the presence of other species of bacteria.     

Minor threonine dehydratase encoded within the threonine synthetic region of Bacillus subtilis

Challenging auxotrophs on metabolites that are precursors of a biosynthetic step involving a mutated enzyme has revealed a new class of suppressor mutations which act by derepressing a minor enzyme activity not normally detected in the wild-type strain. These indirect, partial suppressor mutations which allow isoleucine auxotrophs to grow on homoserine or threonine have been analyzed to determine their effect on enzymes involved in the biosynthesis of these amino acids. It has been found that one class of these suppressor mutations (sprA) leads to the derepression of homoserine kinase, homoserine dehydrogenase, and a minor threonine dehydratase that is not sufficiently active to be detected in the wild-type strain. The gene encoding this second threonine dehydratase activity has been found to be located between the structural genes for homoserine kinase and homoserine dehydrogenase. The results of these experiments indicate that plating of auxotrophs on precursors of a biosynthetic step involving mutated enzymes could prove to be a valuable method for the detection of regulatory mutants as well as a possible tool in studying the evolution of biochemical pathways.