Interaction of aspartate and aspartate-derived antimetabolites with the enzymes of the threonine biosynthetic pathway of Escherichia coli

The five enzymes responsible for the conversion of L-aspartate to L-threonine in Escherichia coli were purified to homogeneity and subsequently reconstituted in vitro in ratios approximating those found in vivo. 31P NMR was used to conveniently monitor the rates of consumption of the substrates ATP and NADPH, the accumulation of the intermediates beta-aspartyl phosphate and homoserine phosphate, and the formation of the products ADP, NADP+, and Pi in a single experiment. By this method, the flux of aspartic acid through the enzymes of the pathway was monitored in the absence and in the presence of several alternative substrates and inhibitors. Several known antimetabolites were found to be alternative substrates that ultimately became inhibitors of pathway flux. L-threo-3-Hydroxyaspartic acid was converted to 3-hydroxyhomoserine phosphate by the first four enzymes of the pathway. The antimetabolite L-threo-3-hydroxyhomoserine was found to bind to and inhibit aspartokinase-homoserine dehydrogenase I in a cooperative fashion (I 0.5 = 3 mM, nH = 2.5), similar to the action of the allosteric end product inhibitor L-threonine (I 0.5 = 0.36 mM, nH = 2.4). In the presence of the remaining enzymes of the pathway, however, L-threo-3-hydroxyhomoserine was phosphorylated to the apparent ultimate antimetabolite L-threo-3-hydroxyhomoserine phosphate that was a potent inhibitor of threonine synthase and consequently of L-threonine biosynthesis. When aspartic acid alone was examined as a substrate of the enzymes of the pathway, no accumulation of the beta-aspartyl phosphate and homoserine phosphate intermediates was observed. However, in the presence of either 5 mM L-threo-3-hydroxyhomoserine or 5 mM L-threo-3-hydroxyhomoserine phosphate, homoserine phosphate was found to accumulate. In contrast to the homoserine phosphate and 3-hydroxyhomoserine phosphate intermediates, both of which were very stable, the acylphosphate intermediates beta-aspartyl phosphate and beta-3-hydroxyaspartyl phosphate were highly susceptible to hydrolysis, with first-order rate constants of 4.6 X 10(-3) min-1 and 4.5 X 10(-2) min-1 (pH 7.8, 25 degrees C), respectively.     

Regulation of lysine biosynthesis in Escherichia coli K12.

A general survey of the regulation in lysine biosynthesis in Escherichia coli K12 is presented. No polygenic operon exists for the genes that code for enzymes of the lysine biosynthetic pathway. Lysyl-tRNA is not directly involved as a co-repressor in the pathway. Different regulation mechanisms must exist for the different enzymes. In the case of the last enzyme, diaminopimelate decarboxylase, its synthesis is induced in vivo by the lysine-sensitive aspartokinase under its non-inhibited allosteric conformation.     

Cloning and expression in Escherichia coli of genes involved in the lysine pathway of Brevibacterium lactofermentum.

The Brevibacterium lactofermentum genes which complement Escherichia coli lysA and asd-1 mutants were identified, respectively, as a 1.9-kilobase PstI-ClaI fragment and a 2.5-kilobase PstI fragment by cloning into pBR325. Southern blot transfers show hybridization to chromosomal fragments of identical size. The putative B. lactofermentum asd and lysA products are 44 and 48 kilodaltons, respectively.     

Role of lysyl-tRNA in the regulation of lysine biosynthesis in Escherichia coli K12.

A mutant of lysyl-tRNA synthetase has been isolated in Escherichia coli K12. With this strain the Kmapp for lysine is 25 fold higher than with the parental strain. The percentage of charged tRNAlys in vivo is only 7 per cent (as against 65 per cent with HFR H). Under these conditions no derepression of synthesis is observed for three lysine biosynthetic enzymes (AK III, ASA-dehydrogenase, DAP-decarboxylase) ; a partial derepression is obtained in the case of the dhdp-reductase. Thus lysyl-tRNA does not act as the only corepressor molecule in the lysine regulon.     

Molecular cloning of menaquinone biosynthetic genes of Escherichia coli K12

Summary A tranducing phage carrying some of the genes (men) defining the early stages of menaquinone biosynthesis was isolated from a pool of recombinant lambda phages that had been constructed from R.HindIII digests of E. coli DNA and the corresponding insertion vector. The lesions of menB and menC mutants were complemented by the phage but menD mutants were transduced either at low frequencies or not at all. This indicates that the transducing phage contains functional menB and menC genes but that only part of the menD gene had been cloned. The phage (G68) was accordingly disignated menCB(D). Studies with the transducing phage enabled earlier mapping data (Guest 1979) to be reinterpreted in favour of the gene order nalA.... menC..menB..menD.... purF. Restriction analyses established the presence of a bacterial DNA fragment (11.5 kb) linked by a R.HindIII target to the right arm of the genome but fused to the left arm of the vector. Hybridization studies confirmed that the cloned DNA was derived from a larger R.HindIII fragment (21 kb). A physical map of the men region was constructed and some flanking and overlapping fragments were identified.     

Regulation of the lysine biosynthetic pathway in Escherichia coli K-12: isolation of a cis-dominant constitutive mutant for AK III synthesis.

A method for isolating regulatory mutants for the synthesis of lysine biosynthetic enzymes in Escherichia coli is described. One of them is identified as a cis-dominant constitutive mutant for the synthesis of the lysine-sensitive asportokinase AK III (lysC gene).     

A relationship between L-serine degradation and methionine biosynthesis in Escherichia coli K12

While wild-type Escherichia coli K12 cannot grow with L-serine as carbon source, two types of mutants with altered methionine metabolism can. The first type, metJ mutants, in which the methionine biosynthetic enzymes are expressed constitutively, are able to grow with L-serine as carbon source. Furthermore, a plasmid carrying the metC gene confers ability to grow on L-serine. These observations suggest that in these mutants, L-serine deamination may be a result of a side-reaction of the metC gene product, cystathionine beta-lyase. The second type is exemplified by two newly isolated strains carrying mutations mapping between 89.6 and 90 min. These mutants use L-serine as carbon source, and also require methionine for growth with glucose at 37 degrees C and above. The phenotypes of the new mutants resemble those of both met and his constitutive mutants in some respects, but have been differentiated from both of them.     

Cloning, expression, purification, and characterization of biosynthetic threonine deaminase from Escherichia coli

Feedback inhibition of the regulatory enzyme threonine deaminase by isoleucine provides an important level of enzymic control over branched chain amino acid biosynthesis in Escherichia coli. Cloning ilvA, the structural gene for threonine deaminase, under control of the trc promoter results in expression of active enzyme upon induction by isopropyl 1-thio-beta-D-galactoside to levels of approximately 20% of the soluble protein in cell extracts. High level expression of threonine deaminase has facilitated the development of a rapid and efficient protocol for the purification of gram quantities of enzyme with a specific activity 3-fold greater than previous preparations. The catalytic activity of threonine deaminase is absolutely dependent on the presence of pyridoxal phosphate, and the tetrameric molecule is isolated containing 1 mol of cofactor/56,000-Da chain. Wild-type threonine deaminase demonstrates a sigmoidal dependence of initial velocity on threonine concentration in the absence of isoleucine, consistent with a substrate-promoted conversion of the enzyme from a low activity to a high activity conformation. The enzymic dehydration of threonine to alpha-ketobutyrate measured by steady-state kinetics, performed at 20 degrees C in 0.05 M potassium phosphate, pH 7.5, is described by a Hill coefficient, nH, of 2.3 and a K0.5 of 8.0 mM. The negative allosteric effector L-isoleucine strongly inhibits the enzyme, yielding a value for nH of 3.9 and K0.5 of 74 mM whereas enzyme activity is greatly increased by L-valine, which yields nearly hyperbolic kinetics characterized by a value for nH of 1.0 and a K0.5 of 5.7 mM. Thus, these effectors promote dramatic and opposing effects on the transition from the low activity to the high activity conformation of the tetrameric enzyme.     

Operator-constitutive mutants in the threonine operon of Escherichia coli K-12.

Three Escherichia coli K-12 mutant strains resistant to DL-alpha-amino-beta-hydroxyvaleric acid were isolated in which the expression of the thr operon is constitutive. The localization and dominance properties of the mutations involved, called thrO, are those of operator mutations. The gene sequence is OABC as suggested by earlier studies.     

The nucleotide sequence recognized by the Escherichia coli K12 restriction and modification enzymes.

The sites recognized by the Escherichia coli K12 restriction endonuclease were localized to defined regions on the genomes of phage φXsK1, φXsK2, and G4 by the marker rescue technique. Methyl groups placed on the genome of plasmid pBR322 by the E. coli K12 modification methylase were mapped in HinfI fragments 1 and 3, and HaeIII fragments 1 and 3. A homology of seven nucleotides in the configuration: 5′-A-A-C .. 6N .. G-T-G-C-3′, where 6N represents six unspecified nucleotides, was found among the DNA sequences containing the five EcoK sites of φXsK1, φXsK2, G4, and pBR322. Three lines of evidence indicate that this sequence constitutes the recognition site of the E. coli K12 restriction enzyme. The C in 5′-A-A-C and the T in 5′-G-T-G-C are locations of mutations leading to loss or gain of the site and thus are positions recognized by the enzyme. This sequence does not occur on φXam3cs70, simian virus 40 (SV40), and fd DNAs which do not possess EcoK sites, and occurs only once on φXsK1, φXsK2, and G4 DNAs, and twice on pBR322 DNA. In order to prove that all seven conserved nucleotides are essential for the recognition by the E. coli K12 restriction enzyme, the nucleotide sequences of φX174, G4, SV40, fd, and pBR322 were searched for sequences differing from the sequence 5′-A-A-C .. 6N .. G-TG-C-3′ at only one of the specified positions. It was found that sequences differing at each of the specified positions occur on DNA sequences that do not contain the EcoK sites. Thus, the recognition site of the E. coli K12 restriction enzyme has the same basic structure as that of the EcoB site (Lautenberger et al., 1978). In each case there are two domains, one containing three and the other four specific nucleotides, separated by a sequence of unspecified bases. However, the unspecified sequence in the EcoK site must be precisely six bases instead of the eight found in the EcoB site. Alignment of the EcoK and EcoB sites suggests that four of the seven specified nucleotides are conserved between the sequences recognized by these two allelic restriction and modification systems.