Aspartokinase isoenzymes of the fruiting myxobacterium Myxoccus xanthus.

Two isoenzymes of aspartokinase can be found in extracts of the differentiating bacterium Myxococcus xanthus. Aspartokinase I is repressed by L-lysine and feedback is inhibited by meso-diaminopimelate and by low concentrations of L-lysine. However, the inhibition by L-lysine is no longer observed at high concentration of this amino acid. Aspartokinase II is repressed and feedback inhibited specifically by L-threonine. Both enzymes are stimulated significantly by L-methionine and L-isoleucine; the effect is greater with aspartokinase I. The role of these enzymes in relation to growth conditions of the organism is discussed and a correlation with life cycle activity is indicated.     

Aspartokinase of Streptococcus mutans: purification, properties, and regulation.

Aspartokinase from Streptococcus mutans BHT was purified to homogeneity and characterized. The molecular weight of the native enzyme was estimated to be 242,000 by gel filtration. Cross-linking of aspartokinase with dimethyl suberimidate and polyacrylamide gel electrophoresis of the amidinated enzyme in the presence of sodium dodecyl sulfate showed the enzyme to be composed of six identical subunits with a molecular wieght of 40,000. The optimal pH range for enzyme activity was 6.5 to 8.5. The apparent Michaelis-Menten constants for aspartate and ATP were 5.5 and 2.2 mM, respectively. The enzyme was stable within the temperature range of 10 to 35 degrees C. Aspartokinase was not feedback inhibited by individual amino acids, but was concertedly inhibited by L-lysine and L-threonine (93.5% inhibition at 10 mM each). The inhibition was noncompetitive with respect to aspartate (Ki = 10 mM) and mixed with respect to ATP. L-Threonine methyl ester and L-threonine amide were able to substitute for L-threonine in feedback inhibition, but the requirement for L-lysine uas strict. The feedback inhibitor pair protected the enzyme against heat denaturation. Aspartokinase synthesis was repressed by L-threonine; this repression was enhanced by L-lysine, but was slightly attenuated by L-methionine.     

Aspartokinase of a lysine producing mutant ofMicrococcus glutamicus

Aspartokinase fromMicrococcus glutamicus AEC RN-13-6/1 [a homoserine requiring, S-(2-aminoethyl)-L-cysteine resistant, lysine producing strain] was purified 71 fold. The partially purified enzyme was inhibited by L-lysine. L-threonine, L-methionine, L-isoleucine, L-valine and L-phenylalanine activated the enzyme and reversed the inhibition by L-lysine. Aspartokinase activity was not derepressed by growth-limiting concentrations of L-threonine and/or L-methionine. It was not repressed by an excess of L-lysine (20 mM) and/or L-isoleucine (15.3 mM). The degree of activation or inhibition by amino acids was dependant on the composition of the growth medium. This observation is in contrast with the enzyme from the original (non-lysine-producing) strain which was inhibited by lysine or threonine and in a concerted manner by threonine plus lysine.     

Unbalance of L-lysine flux in Corynebacterium glutamicum and its use for the isolation of excretion-defective mutants.

We found that the simple addition of L-methionine to the wild type of Corynebacterium glutamicum results in excretion of the cellular building block L-lysine up to rates of 2.5 nmol/min/mg (dry weight). Biochemical analyses revealed that L-methionine represses the homoserine dehydrogenase activity and reduces the intracellular L-threonine level from 7 to less than 2 mM. Since L-lysine synthesis is regulated mainly by L-threonine (plus L-lysine) availability, the result is enhanced flux towards L-lysine. This indicates a delicate and not well controlled type of flux control at the branch point of aspartate semialdehyde conversion to either L-lysine or L-threonine, probably due to the absence of isoenzymes in C. glutamicum. The inducible system of L-lysine excretion discovered was used to isolate mutants defective in the excretion of this amino acid. One such mutant characterized in detail accumulated 174 mM L-lysine in its cytosol without extracellular excretion of L-lysine, whereas the wild type accumulated 53 mM L-lysine in the cytosol and 5.9 mM L-lysine in the medium. The mutant was unaffected in L-lysine uptake or L-isoleucine or L-glutamate excretion, and also the membrane potential was unaltered. This mutant therefore represents a strain with a defect in an excretion system for the primary metabolite L-lysine.     

Regulation of aspartokinase and homoserine dehydrogenase in acetic acid bacteria

The regulation of aspartokinase and homoserine dehydrogenase has been studied in three Acetobacter and two Gluconobacter species. Both enzymes were regulated by feedback inhibition. Aspartokinase was inhibited by L-threonine and concertedly inhibited by L-threonine plus L-lysine. The homoserine dehydrogenase was NADP-specific and was inhibited by L-threonine. Separation of the two enzymes by ammonium sulphate fractionation was possible in Acetobacter peroxydans, A. rancens and Gluconobacter melanogenus but not in A. liquefaciens or G. oxydans.     

Metabolism of aspartate in Mycobacterium smegmatis

Mycobacterium smegmatis grows best on L-asparagine as a sole nitrogen source; this was confirmed. [14C]Aspartate was taken up rapidly (46 nmol.mg dry cells-1.h-1 from 1 mM L-asparagine) and metabolised to CO2 as well as to amino acids synthesised through the aspartate pathway. Proportionately more radioactivity appeared in the amino acids in bacteria grown in medium containing low nitrogen. Activities of aspartokinase and homoserine dehydrogenase, the initial enzymes of the aspartate pathway, were carried by separate proteins. Aspartokinase was purified as three isoenzymes and represented up to 8% of the soluble protein of M. smegmatis. All three isoenzymes contained molecular mass subunits of 50 kDa and 11 kDa which showed no activity individually; full enzyme activity was recovered on pooling the subunits. Km values for aspartate were: aspartokinases I and III, 2.4 mM; aspartokinase II, 6.4 mM. Aspartokinase I was inhibited by threonine and homoserine and aspartokinase III by lysine, but aspartokinase II was not inhibited by any amino acids. Aspartokinase activity was repressed by methionine and lysine with a small residue of activity attributable to unrepressed aspartokinase I. Homoserine dehydrogenase activity was 96% inhibited by 2 mM threonine; isoleucine, cysteine and valine had lesser effects and in combination gave additive inhibition. Homoserine dehydrogenase was repressed by threonine and leucine. Only amino acids synthesised through the aspartate pathway were tested for inhibition and repression. Of these, only one, meso-diaminopimilate, had no discernable effect on either enzyme activity.     

Construction of Lactobacillus plantarum strain with enhanced L-lysine yield

AIM: To enhance L-lysine secretion in Lactobacillus plantarum. METHODS AND RESULTS: An S-2-aminoethyl-L-cystein (AEC)-resistant mutant of L. plantarum was isolated, and it produced L-lysine at considerably higher level than the parent strain. Aspartokinase in the mutant has been desensitized to feedback inhibition by L-lysine. The nucleotide sequence analysis of thrA2 that codes for aspartokinase in the mutant predicted a substitution of glutamine to histidine at position 421. L-Lysine-insensitive aspartokinase, together with aspartate semialdehyde dehydrogenase, dihydrodipicolinate synthase, and dihydrodipicolinate reductase genes, was cloned from L. plantarum DNA to a shuttle vector, pRN14, and the genes were then transformed individually into the AEC-resistant mutant and the parent strain. The overexpression of the genes led to the increase in the activity of enzymes they encode in vitro. However, only the strain overexpressing aspartokinase or dihydrodipicolinate synthase produced more L-lysine. CONCLUSIONS: The desensitization of aspartokinase to L-lysine in L. plantarum led to the overproduction of L-lysine. The overexpression of L-lysine-insensitive aspartokinase or dihydrodipicolinate synthase enhanced L-lysine secretion in L. plantarum. SIGNIFICANCE AND IMPACT OF THE STUDY: The use of the L-lysine-overproducing strain of L. plantarum in food or feed fermentation may increase the L-lysine content of fermented products.     

The threonine-sensitive homoserine dehydrogenase and aspartokinase activities of Escherichia coli K-12. Incubation of the enzyme in alkaline conditions: dissociation and disulfide-bridge formation.

Aspartokinase I - homoserine dehydrogenase I from Escherichia coli K-12, a homotetrameric enzyme, dissociates into dimers upon alkaline treatment. Both aspartokinase and homoserine dehydrogenase inactivation, as well as desensitazion towards L-threonine, occur in a multi-step process. Dithiothreitol stabilizes a dimeric form retaining full activity and sensitivity; L-homoserine stabilizing another dimeric form devoid of aspartokinase activity and retaining a substantial dehydrogenase activity insensitive toward L-threonine. A model is proposed showing that dissociation into dimers occurs in a first step, the resulting dimer losing both aspartokinase and homoserine dehydrogenase sensitivity in two subsequent steps involving the formation of intrachain disulfide bonds.     

Expression of the gene for Bacillus subtilis aspartokinase II in Escherichia coli

The gene coding for the subunits of aspartokinase II from Bacillus subtilis has been identified in a B. subtilis DNA library and cloned in a bacterial plasmid (Bondaryk, R. P., and Paulus, H. (1984) J. Biol. Chem. 259, 585-591). The introduction of a plasmid carrying the aspartokinase II gene into an auxotrophic Escherichia coli strain lacking all three aspartokinases restored its ability to grow in the absence of L-lysine, L-threonine, and L-methionine. The B. subtilis aspartokinase gene could thus be functionally expressed in E. coli and substitute for the E. coli aspartokinases. Measurement of aspartokinase levels in extracts of aspartokinaseless E. coli transformed with the B. subtilis aspartokinase II gene revealed an enzyme level comparable to that in a genetically derepressed B. subtilis strain. In spite of the high level of aspartokinase, the growth of the transformed E. coli strain was severely inhibited by the addition of L-lysine but could be restored by also adding L-homoserine. This apparently paradoxical sensitivity to lysine was due to the allosteric inhibition of B. subtilis aspartokinase II by that amino acid, a property which was also observed in extracts of the transformed E. coli strain. The synthesis and degradation of the aspartokinase II subunits were measured by labeling experiments in E. coli transformed with the B. subtilis aspartokinase II gene. In contrast to exponentially growing cells of B. subtilis which contained equimolar amounts of the aspartokinase alpha and beta subunits, the transformed E. coli strain contained a 3-fold molar excess of beta subunit. Pulse-chase experiments showed that the disproportionate level of beta subunit was not due to more rapid turnover of alpha subunit, both subunits being quite stable, but presumably to a more rapid rate of synthesis. After the addition of rifampicin, the synthesis of alpha subunit declined much more rapidly than that of beta subunit, indicating that the two subunits were translated independently from mRNA species that differ in functional stability. In conjunction with the results described in the preceding paper which demonstrated that the aspartokinase subunits are encoded by a single DNA sequence, these observations imply that the alpha and beta subunits of B. subtilis aspartokinase II are the products of in-phase overlapping genes.     

Sequence analysis and expression of the aspartokinase and aspartate semialdehyde dehydrogenase operon from rifamycin SV-producing amycolatopsis mediterranei.

A approximately 4.8 kb KpnI fragment, from the upstream region of the methylmalonyl-CoA mutase gene (mutAB) of rifamycin SV-producing Amycolatopsis mediterranei, was cloned and partially sequenced. Codon preference analysis showed three complete ORFs. ORF2 is internal to ORF1, and encodes a polypeptide corresponding to 172 amino acids, whereas ORF1 encodes a polypeptide of 421 amino acids. They were identified as the encoding genes of aspartokinase alpha- and beta-subunits by comparing the amino acid sequences with those in the database. The downstream ORF3, whose start codon was overlapped with the stop codon of both ORF1 and ORF2 by 1 bp, was identified as the aspartate semialdehyde dehydrogenase gene (asd), encoding a polypeptide of 346 amino acids. Subclones containing either the ask gene or the asd gene were constructed, in which the genes could be expressed under Lac promoters. Two subclones could transform E. coli CGSC 5074 (ask-) and E. coli X6118 (asd-) to prototrophy, supporting the functional assignments. Southern hybridisation indicated that the approximately 4.8 kb sequenced region represented a continuous segment in the A. mediterranei chromosome. It is concluded that ask and asd genes are present in an operon in A. mediterranei, and therefore that organisation of these two genes is the same as in most gram-positive bacteria, such as Mycobacteria, Corynebacterium glutamicum and Bacillus subtilis, but is different from Streptomyces akiyoshiensis.     

Effects of a feedback-resistant aspartokinase III gene on L-isoleucine production in Escherichia coli K-12

An L-isoleucine-overproducing recombinant strain of E. coli, TVD5, was also found to overproduce L-valine. The L-isoleucine productivity of TVD5 was markedly decreased by addition of L-lysine to the medium. Introduction of a gene encoding feedback-resistant aspartokinase III increased L-isoleucine productivity and decreased L-valine by-production. The resulting strain accumulated 12 g/l L-isoleucine from 40 g/l glucose, and suppression of L-isoleucine productivity by L-lysine was relieved.     

Activities and regulation of the enzymes involved in the first and the third steps of the aspartate biosynthetic pathway in Enterococcus faecium

The enzymes aspartokinase and homoserine dehydrogenase catalyze the reaction at key branching points in the aspartate pathway of amino acid biosynthesis. Enterococcus faecium has been found to contain two distinct aspartokinases and a single homoserine dehydrogenase. Aspartokinase isozymes eluted on gel filtration chromatography at molecular weights greater than 250,000 and about 125,000. The molecular weight of homoserine dehydrogenase was determined to be 220,000. One aspartokinase isozyme was slightly inhibited by meso-diaminopimelic acid. Another aspartokinase was repressed and inhibited by lysine. Although the level of diaminopimelate-sensitive (DAP^s) enzyme was not much affected by growth conditions, the activity of lysine-sensitive (Lys^s) aspartokinase disappeared rapidly during the stationary phase and was depressed in rich media. The synthesis of homoserine dehydrogenase was controlled by threonine and methionine. Threonine also inhibited the specific activity of this enzyme. The regulatory properties of aspartokinase isozymes and homoserine dehydrogenase from E. faecium are discussed and compared with those from Bacillus subtilis.     

Production and characterization of bifunctional enzymes. Substrate channeling in the aspartate pathway

The direct channeling of an intermediate between enzymes that catalyze consecutive reactions in a pathway offers the possibility of an efficient, exclusive, and protected means of metabolite delivery. Aspartokinase-homoserine dehydrogenase I (AK-HDH I) from Escherichia coli is an unusual bifunctional enzyme in that it does not catalyze consecutive reactions. The potential channeling of the intermediate beta-aspartyl phosphate between the aspartokinase of this bifunctional enzyme and aspartate semialdehyde dehydrogenase (ASADH), the enzyme that catalyzes the intervening reaction, has been examined. The introduction of increasing levels of inactivated ASADH has been shown to compete against enzyme-enzyme interactions and direct intermediate channeling, leading to a decrease in the overall reaction flux through these consecutive enzymes. These same results are obtained whether these experiments are conducted with aspartokinase III, a naturally occurring monofunctional isozyme, with an artificially produced monofunctional aspartokinase I, or with a fusion construct of AK I-ASADH. These results provide definitive evidence for the channeling of beta-aspartyl phosphate between aspartokinase and aspartate semialdehyde dehydrogenase in E. coli and suggest that ASADH may provide a bridge to channel the intermediates between the non-consecutive reactions of AK-HDH I.     

Production and characterization of bifunctional enzymes. Domain swapping to produce new bifunctional enzymes in the aspartate pathway

The bifunctional enzyme aspartokinase-homoserine dehydrogenase I from Escherichia coli catalyzes non-consecutive reactions in the aspartate pathway of amino acid biosynthesis. Both catalytic activities are subject to allosteric regulation by the end product amino acid L-threonine. To examine the kinetics and regulation of the enzymes in this pathway, each of these catalytic domains were separately expressed and purified. The separated catalytic domains remain active, with each of their catalytic activities enhanced in comparison to the native enzyme. The allosteric regulation of the kinase activity is lost, and regulation of the dehydrogenase activity is dramatically decreased in these separate domains. To create a new bifunctional enzyme that can catalyze consecutive metabolic reactions, the aspartokinase I domain was fused to the enzyme that catalyzes the intervening reaction in the pathway, aspartate semialdehyde dehydrogenase. A hybrid bifunctional enzyme was also created between the native monofunctional aspartokinase III, an allosteric enzyme regulated by lysine, and the catalytic domain of homoserine dehydrogenase I with its regulatory interface domain still attached. In this hybrid the kinase activity remains sensitive to lysine, while the dehydrogenase activity is now regulated by both threonine and lysine. The dehydrogenase domain is less thermally stable than the kinase domain and becomes further destabilized upon removal of the regulatory domain. The more stable aspartokinase III is further stabilized against thermal denaturation in the hybrid bifunctional enzyme and was found to retain some catalytic activity even at temperatures approaching 100 degrees C.     

Mutation analysis of the feedback inhibition site of aspartokinase III of Escherichia coli K-12 and its use in L-threonine production.

Aspartokinase III (AKIII), one of three isozymes of Escherichia coli K-12, is inhibited allosterically by L-lysine. This enzyme is encoded by the lysC gene and has 449 amino acid residues. We analyzed the feedback inhibition site of AKIII by generating various lysC mutants in a plasmid vector. These mutants conferred resistance to L-lysine and/or an L-lysine analogue on their host. The inhibitory effects of L-lysine on and heat tolerance of 14 mutant enzymes were examined and DNA sequencing showed that the types of mutants were 12. Two hot spots, amino acid residue positions 318-325 and 345-352, were detected in the C-terminal region of AKIII and these enzyme regions may be important in L-lysine-mediated feedback inhibition of AKIII. Feedback resistant lysC relieved on L-threonine hyper-producing strain, B-3996, from reduced L-threonine productivity by addition of L-lysine, and furthermore increased L-threonine productivity even when no addition of L-lysine. It suggested that the bottleneck of L-threonine production of B-3996 was AK and feedback resistant lysC was effective because of the strict inhibition by cytoplasmic L-lysine.     

Characterization of the L-lysine biosynthetic pathway in the obligate methylotroph Methylophilus methylotrophus

The L-lysine biosynthetic pathway of the gram-negative obligate methylotroph Methylophilus methylotrophus AS1 was examined through characterization of the enzymes aspartokinase (AK), aspartsemialdehyde dehydrogenase, dihydrodipicolinate synthase (DDPS), dihydrodipicolinate reductase, and diaminopimelate decarboxylase. The AK was inhibited by L-threonine and by a combination of L-threonine and L-lysine, but not by L-lysine alone, and the activity of DDPS was moderately reduced by L-lysine. In an L-lysine producing mutant (G49), isolated as an S-(2-aminoethyl)-L-cysteine (lysine analog) resistant strain, both AK and DDPS were partially resistant to feedback inhibition. The ask and dapA genes encoding AK and DDPS respectively were isolated from the parental strain, AS1, and its G49 derivative. Comparison of the sequences revealed a point mutation in each of these genes in G49. The mutation in the ask gene altered aspartic acid in a key region involved in the allosteric regulation common to AKs, while a novel mutation in the dapA gene altered tyrosine-106, which was assumed to be involved in the binding of L-lysine to DDPS.     

Regulation of lysine and threonine synthesis in carrot cell suspension cultures and whole carrot roots

Aspartokinase (EC 2.7.2.4), homoserine-dehydrogenase (EC 1.1.1.3) and dihydrodipicolinic-acid-synthase (EC 4.2.1.52) activities were examined in extracts from 1-year-old and 11-year-old cell suspension cultures and whole roots of garden carrot (Daucus carota L.). Aspartokinase activity from suspension cultures was inhibited 85% by 10 mM L-lysine and 15% by 10mM L-threonine. In contrast, aspartokinase activity from whole roots was inhibited 45% by 10 mM lysine and 55% by 10 mM threonine. This difference may be based upon alterations in the ratios of the two forms (lysine-and threonine-sensitive) of aspartokinase, since the activity is consistently inhibited 100% by lysine+threonine. Only one form each of homoserine dehydrogenase and of dihydrodipicolinic acid synthase was found in extracts from cell suspension cultures and whole roots. The regulatory properties of either enzyme were identical from the two sources. In both the direction of homoserine formation and aspartic--semialdehyde formation, homoserine dehydrogenase activities were inhibited by 10mM threonine and 10 mM L-cysteine in the presence of NADH or NADPH. KCl increased homoserine dehydrogenase activity to 185% of control values and increased the inhibitory effect of threonine. Dihydrodipicolinic acid synthase activities from both sources were inhibited over 80% by 0.5 mM lysine. Aspartokinase was less sensitive to inhibition by low concentrations of lysine and threonine than were dihydrodipicolinic acid synthase and homoserine dehydrogenase to inhibition by the respective inhibitors.     

Additional effects of monovalent cations on the lysine-sensitive aspartokinase of E. coli B

The apparent specificity of activation of lysine-sensitive aspartokinase (E.C.2.7.2.4) from E. coli by monovalent cations differs depending on the assay used and on the Mg2+ concentration. Activity is nearly absolutely dependent on and is highly specific for a monovalent cation in the aspartate semialdehyde dehydrogenase coupled assay or the adenosine triphosphate-adenosine diphosphate exchange assay. Little specificity for monovalent cations is observed using the aspartyl hydroxamate assay. Activation and specificity are also altered by Mg2+ concentrations at a constant 5 mM nucleotide concentration. At a low (1.25 or 1.6 mM)Mg2+ concentration, monovalent cation activation and specificity are nearly absolute. Less dependence on monovalent cations and less specificity are observed at a higher Mg2+ concentration (6 mM). Li+ inhibits aspartokinase competitively with respect to either K+ or NH4+. Monovalent cations are also thermoprotective and differential thermal inactivation experiments at 56 degrees C reveal that NH4+ and K+, either of which will produce maximum catalytic activity, interact differently with aspartokinase. K+ interacts with positive cooperativity, whereas NH4+ does not. K+, NH4+, and Na+ are about equally effective in enhancing the dissociation of the aspartokinase-aspartylphosphate complex. Li+ is less effective.     

Identification and expression of a cDNA from Daucus carota encoding a bifunctional aspartokinase-homoserine dehydrogenase

Aspartokinase (EC 2.7.2.4) and homoserine dehydrogenase (EC 1.1.1.3) catalyze steps in the pathway for the synthesis of lysine, threonine, and methionine from aspartate. Homoserine dehydrogenase was purified from carrot (Daucus carota L.) cell cultures and portions of it were subjected to amino acid sequencing. Oligonucleotides deduced from the amino acid sequences were used as primers in a polymerase chain reaction to amplify a DNA fragment using DNA derived from carrot cell culture mRNA as template. The amplification product was radiolabelled and used as a probe to identify cDNA clones from libraries derived from carrot cell culture and root RNA. Two overlapping clones were isolated. Together the cDNA clones delineate a 3089 bp long sequence encompassing an open reading frame encoding 921 amino acids, including the mature protein and a long chloroplast transit peptide. The deduced amino acid sequence has high homology with the Escherichia coli proteins aspartokinase I-homoserine dehydrogenase I and aspartokinase II-homoserine dehydrogenase II. Like the E. coli genes the isolated carrot cDNA appears to encode a bifunctional aspartokinase-homoserine dehydrogenase enzyme.Abbreviations AK aspartokinase - HSDH homoserine dehydrogenase - PCR polymerase chain reaction - SDS sodium dodecyl sulfate The mention of vendor or product does not imply that they are endorsed or recommended by the U.S. Department of Agriculture over vendors of similar products not mentioned.