Mutants of Escherichia coli with a growth requirement for either lysine or pyridoxine

Two distinct phenotypic classes of lysine requiring auxotrophs of Escherichia coli are described. Mutants of the LysA class produce little or no active diaminopimelic acid (DAP) decarboxylase and specifically require lysine for growth. Mutants of the LysB class produce a cryptic DAP decarboxylase which can be activated both in vivo and in vitro by higher than normal levels of its cofactor, pyridoxal 5'-phosphate. The LysB mutants have an alternate requirement for lysine or pyridoxine. Both LysA and LysB mutations map at 55 min, close to the thyA locus of E. coli. The association between pyridoxal phosphate and DAP decarboxylase appears to be much weaker in LysB mutants than in wild-type bacteria, and the mutant enzyme also sediments more slowly than wild-type enzyme in sucrose density gradients. The results suggest that the LysB mutations alter a specific region (or subunit) of the enzyme molecule which is needed to stabilize the binding of pyridoxal phosphate. These studies help to resolve certain contradictory observations on DAP decarboxylase reported earlier and may have relevance to pyridoxal phosphate enzymes in general. Prototrophic revertants of LysB mutants arise by second site mutations that result in increased availability of intracellular pyridoxal phosphate. These revertants appear to be derepressed for pyridoxine biosynthesis.     

Analysis of catalytic determinants of diaminopimelate and ornithine decarboxylases using alternate substrates

Diaminopimelate decarboxylase (DAPDC) and ornithine decarboxylase (ODC) are pyridoxal 5'-phosphate dependent enzymes that are critical to microbial growth and pathogenicity. The latter is the target of drugs that cure African sleeping sickness, while the former is an attractive target for antibacterials. These two enzymes share the (β/α)(8) (i.e., TIM barrel) fold with alanine racemase, another pyridoxal 5'-phosphate dependent enzyme critical to bacterial survival. The active site structural homology between DAPDC and ODC is striking even though DAPDC catalyzes the decarboxylation of a D stereocenter with inversion of configuration and ODC catalyzes the decarboxylation of an L stereocenter with retention of configuration. Here, the structural and mechanistic bases of these interesting properties are explored using reactions of alternate substrates with both enzymes. It is concluded that simple binding determinants do not control the observed stereochemical specificities for decarboxylation, and a concerted decarboxylation/proton transfer at Cα of the D stereocenter of diaminopimelate is a possible mechanism for the observed specificity with DAPDC.     

Regulation of lysine biosynthesis in Brevibacterium flavum

L-lysine synthesis pathway enzyme activities: β-aspartate kinase (EC.2.7.2.4), diaminopimelate decarboxylase (EC.4.1.1.20) for two L-lysine producing strains Brevibacterium flavum 22LD and RC-115 were studied. It has been found that β-aspartate kinase and diaminopimelate decarboxylase in the Br. flavum RC-115 are less sensitive to feed-back inhibition by lysine and threonine. It is supposed that desensitized β-aspartate kinase in the Br. flavum RC-115 can be determined by genetical changes of the regulatory properties of the β-aspartate kinase. Auxotrophity in the locus of homoserine dehydrogenase was tested and no homoserine dehydrogenase (EC.1.1.1.3) activity was found in either strain. The combination of these both types of mutation supplemented by the lack of catabolic repression in the RC-115 strain makes it an active lysine producer in the medium with high carbohydrates content.     

Biochemical characterization of lysine auxotrophs of Staphylococcus aureus

Lysine biosynthesis in Staphylococcus aureus has been studied by use of a series of lysine auxotrophs. The strains were isolated after chemical mutagenesis. The majority of these mutant strains were classified according to the enzymatic step found to be deficient. Specific enzyme assays as well as nutritional tests were used to group the organisms. The enzymes included were dihydrodipicolinate synthetase, dihydrodipicolinate reductase, diaminopimelate epimerase, and diaminopimelate decarboxylase. The accumulation of diaminopimelate in certain mutants and the demonstration of dihydrodipicolinate synthetase and reductase provide the first detailed evidence that S. aureus utilizes the diaminopimelate pathway for lysine biosynthesis. A cell-free system was used to study the regulation of these enzymes with the exception of diaminopimelate epimerase. Lysine repressed all of the enzymes tested. The repression appeared to be coordinate in nature. The data presented provide suggestive evidence that the lysine biosynthetic region in S. aureus constitutes an operon.     

Chemical modification of the lysine residues of bacterial formate dehydrogenase

Inactivation of formate dehydrogenase by formaldehyde, pyridoxal and pyridoxal phosphate was studied. The effects of concentrations of the modifying agents, substrates, products and inhibitors on the extent of the enzyme inactivation were examined. A complete formate dehydrogenase inactivation by pyridoxal, pyridoxal, phosphate and formaldehyde is achieved by the blocking of 2, 5 and 13 lysine residues per enzyme subunit, respectively. The coenzymes do not protect formate dehydrogenase against inactivation. In the case of modification by pyridoxal and pyridoxal phosphate a complete maintenance of the enzyme activity and specific protection of one lysine residue per enzyme subunit is observed during formation of a binary formate-enzyme complex, or a ternary enzyme--NAD--azide complex. One lysine residue is supposed to be located at the formate-binding site of the formate dehydrogenase active center.     

The effect of pyridoxal phosphate on the activity of aldolase from Lemna minor L.

Lemna aldolase has been purified by ion-exchange and affinity chromatography. The enzyme is inhibited by pyridoxal phosphate in a manner which suggests that pyridoxal phosphate forms a non-covalent complex with the enzymes which is in equilibrium with the Schiff base covalently modified enzyme. The kinetics of the reversal of inhibition have been used to test the proposition that the fall in aldolase activity observed during periods of nitrogen starvation is due to inhibition by pyridoxal phosphate. It is concluded that the in vivo loss of aldolase activity is not due to pyridoxal phosphate and that the in vitro inhibition of glycolytic enzymes by pyridoxal phosphate is due to the reaction with lysine residues at the active sites which are necessary to bind the strongly acidic sugar phosphates.     

Control of diaminopimelate decarboxylase by L-lysine during growth and sporulation of Bacilluscereus

l-Lysine caused repression of diaminopimelate decarboxylase synthesis in Bacillus cereus when grown in either a minimal defined medium (CDGS medium) or a complex defined medium (a modified lysine assay medium). When cells were grown in either of the two media, variations in the specific activity of the enzyme as a function of time were found to be correlated with the intracellular lysine pool size during growth. From all of the data presented, it seems reasonable to conclude that during growth the synthesis of diaminopimelate decarboxylase is probably regulated by the intracellular lysine pool size. The relationship between lysine pool concentration and the specific activity of the enzyme did not occur in sporulating cells. The specific activity of diaminopimelate decarboxylase started to decrease at the end of exponential growth and continued to decline until it became nondetectable at the time of dipicolinic acid synthesis and development of spore refractility. Throughout this time, the intracellular lysine pool size remained below that which allowed derepression of enzyme synthesis during exponential growth. The mechanism(s) responsible for the observed decrease in the specific activity of the enzyme at the end of exponential growth is unknown. A threefold rise in the intracellular diaminopimelic acid concentration occurred when there was little or no detectable enzyme activity at the time of dipicolinic acid synthesis. This accumulation of diaminopimelic acid may exert positive control on the synthesis of spore peptidoglycan, the major component of the spore cortex.     

Inactivation of yeast fatty acid synthetase by modifying the beta-ketoacyl reductase active lysine residue with pyridoxal 5'-phosphate

Treatment of yeast fatty acid synthetase with pyridoxal 5'-phosphate inhibited the enzyme. Assays of the partial activities of the pyridoxal phosphate-treated synthetase showed that only the beta-ketoacyl reductase was significantly inhibited. NADPH prevented inactivation of the enzyme by pyridoxal phosphate, indicating that pyridoxal modifies a residue near or in the beta-ketoacyl reductase site. The pyridoxal-treated synthetase shows a fluorescence spectrum with a maximum of 426 nm after uv irradiation at 325 nm. Binding of the pyridoxal phosphate to the synthetase is reversible as shown by the disappearance of the fluorescence band after dialysis of pyridoxal-treated enzyme. Reduction with NaBH4 of the pyridoxal-treated enzyme eliminates this fluorescence maximum and causes the appearance of a new band at 393 nm. These observations suggest that pyridoxal phosphate interacts with the synthetase by forming a Schiff base with lysine residue at the beta-ketoacyl reductase site. Amino acid analyses of the HCl hydrolysates of the borohydride-reduced, pyridoxal-treated synthetase showed the presence of 6 mol of N6-pyridoxal derivative of lysine per mole of fatty acid synthetase, indicating the presence of six sites of beta-ketoacyl reductase in the native enzyme. Autoradiography of sodium dodecyl sulfate-polyacrylamide gels of the pyridoxal phosphate enzyme reduced with NaB3H4 indicates that the alpha subunit contains the beta-ketoacyl reductase domain. These findings are consistent with the proposed structure of the alpha 6 beta 6 complex required for palmitoyl-CoA synthesis.     

Metabolic interlock between the acetolactate synthase isoenzymes and lysine biosynthesis in Escherichia coli K-12

Summary Some of the strains containing mutations in the genes for the acetolactate synthase isoenzymes are temperature sensitive (ts). Suppression of the acetolactate synthase defect due to one of these mutations suppresses also the ts phenotype; moreover, a genetic cross shows that the two phenotypes cannot be dissociated.The ts phenotype is accompanied by a decreased efficiency of transduction with Pl phage. Observations at the light microscope show formation of abnormal cells. Under specific conditions diaminopimelate stimulates growth and restores normal transduction efficiency. The rate of diaminopimelate formed and excreted by non-growing cells decreases when an acetolactate synthase mutation is present.We give evidence that the ts phenotype is due to an increased formation of lysine from diaminopimelate; this causes a starvation for the latter and therefore cell wall abnormalities. In fact, even at the permissive temperature, the lysine pool is 8x increased in a strain with an acetolactate synthase defect, while a slight decrease in the diaminopimelate pool is observed. Moreover, introduction into a ts strain of a mutation in lysA (the gene coding for diaminopimelate decarboxylase) cures the ts phenotype. Finally among the temperature resistant revertants we found some lysine auxotrophs.     

Regulation of enzymes of lysine biosynthesis in Corynebacterium glutamicum

The regulation of the six enzymes responsible for the conversion of aspartate to lysine, together with homoserine dehydrogenase, was studied in Corynebacterium glutamicum. In addition to aspartate kinase activity, the synthesis of diaminopimelate decarboxylase was also found to be regulated. The specific activity of this enzyme was reduced to one-third in extracts of cells grown in the presence of lysine. Aspartate-semialdehyde dehydrogenase, dihydrodipicolinate synthase, dihydrodipicolinate reductase, and diaminopimelate dehydrogenase were neither influenced in their specific activity, nor inhibited, by any of the aspartate family of amino acids. Homoserine dehydrogenase was repressed by methionine (to 15% of its original activity) and inhibited by threonine (4% remaining activity). Inclusion of leucine in the growth medium resulted in a twofold increase of homoserine dehydrogenase specific activity. The flow of aspartate semialdehyde to either lysine or homoserine was influenced by the activity of homoserine dehydrogenase or dihydrodipicolinate synthase. Thus, the twofold increase in homoserine dehydrogenase activity resulted in a decrease in lysine formation accompanied by the formation of isoleucine. In contrast, repression of homoserine dehydrogenase resulted in increased lysine formation. A similar increase of the flow of aspartate semialdehyde to lysine was found in strains with increased dihydrodipicolinate synthase activity, constructed by introducing the dapA gene of Escherichia coli (coding for the synthase) into C. glutamicum.     

Lysine biosynthesis inMethanobacterium thermoautotrophicum is by the diaminopimelic acid pathway

Methanobacterium thermoautotrophicum, an archaebacterium, possesses the first and last enzymes of the diaminopimelic acid pathway for lysine biosynthesis, dihydrodipicolinate synthase, and diaminopimelate decarboxylase. It does not have saccharopine dehydrogenase, the last enzyme of the aminoadipate pathway for lysine biosynthesis. The dihydrodipicolinate synthase is inhibited but not repressed by lysine. We conclude that this microbe uses the diaminopimelate pathway for synthesis of lysine.Deceased.     

Modification of pyruvate, phosphate dikinase with pyridoxal 5'-phosphate: evidence for a catalytically critical lysine residue

Pyruvate, phosphate dikinase from Bacteroides symbiosus is strongly inhibited by low concentrations of pyridoxal 5'-phosphate. The inactivation follows pseudo-first-order kinetics over an inhibitor concentration range of 0.1-2 mM. The inactivation is highly specific since pyridoxine and pyridoxamine 5'-phosphate, analogues of pyridoxal 5'-phosphate, which lack an aldehyde group, caused little or no inhibition even at high concentrations. The unreduced dikinase-pyridoxal 5'-phosphate complex displays an absorption maxima near 420 nm, typical for Schiff base formation. Following reduction of the Schiff base with sodium borohydride, N6-pyridoxyllysine was identified in the acid hydrolysate. When the enzyme was incubated in the presence of pyridoxal 5'-phosphate and reducing agent, the ATP/AMP, Pi/PPi, and pyruvate/phosphoenolpyruvate isotopic exchange reactions were inhibited to approximately the same extent, suggesting that the modification of the lysyl moiety causes changes in the enzyme that affect the reactivity of the pivotal histidyl residue. Phosphorylation of the histidyl group appears to prevent the inhibitor from attacking the lysine residue. On the other hand, addition of pyridoxal 5'-phosphate to the pyrophosphorylated enzyme promotes release of the pyrophosphate and yields the free enzyme which is subject to inhibition.     

Effect of mutations affecting lysyl-tRNAlys on the regulation of lysine biosynthesis in Escherichia coli.

Summary When studying mutants affecting lysyl-tRNA synthetase or tRNA^Lys (hisT, hisW), a lack of correlation is clearly observed between the amount of lysyl-tRNA and the level of derepression of several lysine biosynthetic enzymes. This excludes the possible role of lysyl-tRNA as the specific corepressor of the lysine regulon. However, the level of derepression of DAP-decarboxylase, the last enzyme of the lysine pathway, is very low in the hisT mutant; this indicates that tRNA^Lys is a secondary effector involved in the regulation of the synthesis of this enzyme.Abbreviations DAP diaminopimelate - KRS lysyl-tRNA synthetase - L-lysine tRNA ligase (AMP) (EC6.1.16) - AK III lysinesensitive aspartokinase (EC 2.7.24) - ASA-dehydrogenase aspartic semialdehyde dehydrogenase (EC 1.2.1.10) - DHDP-reductase dihydrodipicolinic acid reductase - DAP-decarboxylase diaminopimelate decarboxylase (EC 4.1.1.20) - AK I threonine-sensitive aspartokinase - HDHI threonine-sensitive homoserine dehydrogenase     

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.     

Lysine production byBrevibacterium linens and its mutants: Activities and regulation of enzymes of the lysine biosynthetic pathway

Activity and regulation of key enzymes of the lysine biosynthetic pathway were investigated inBrevibacterium linens, a natural excretor of lysine, its lysine-overproducing homoserine auxotroph (Hom(-1)) and its auxotrophic and multianalogue-resistant high-yielding mutant (AEC NV 20(r)50). The activity of aspartate kinase (AK) and aspartaldehydate dehydrogenase (AD) was maximum during the mid-exponential phase of growth and decreased therafter. The mutants showed 10 and 20% more activity of AK and AD than the wild-type lysine excretor.B. linens (natural excretor) has a single AK and AD repressed and inhibited bivalently by lysine and threonine. Lysine slightly repressed and inhibited dihydrodipicolinate synthase (DS) and diaminopimelate decarboxylase (DD) of the wild type and of the mutant Hom(-1). The mutant AEC NV 20(r)50 showed DS and DD to be insensitive to lysine inhibition and repression. Persistence of a major part of the maximal activity of these enzymes during the late stationary phase of growth allowed prolonged synthesis and excretion of lysine. Stepwise addition of resistance to the different analogues of lysine in the mutant AEC NV20(r)50 resulted in an increase of enzyme activity and reduced repressibilities of enzymes that contributed to the high yield of lysine.     

Activation by adenosine-5'-triphosphate of glutamic decarboxylase from a subcellular fraction of mouse brain.

Glutamate decarboxylase from a mouse brain P2 fraction undergoes a twofold activation in the presence of 0.5 mM ATP. No such stimulation by ATP occurs if the enzyme is assayed in the presence of excess pyridoxal phosphate as cofactor. The ATP-induced stimulation is almost completely eliminated if the enzyme is dialysed before its assay. [lambda-32P]ATP present during the enzyme measurement is converted to [32P]pyridoxal phosphate. These results demonstrate that the activation produced by ATP is the result of the generation of cofactor during the course of the assay. This phenomenon may be a reflection of a control mechanism of glutamate decarboxylase activity.     

Horse liver alcohol dehydrogenase. A study of the essential lysine residue.

1. The inactivation of horse liver alcohol dehydrogenase by pyridoxal 5'-phosphate in phosphate buffer, pH8, at 10 degrees C was investigated. Activity declines to a minimum value determined by the pyridoxal 5'-phosphate concentration. The maximum inactivation in a single treatment is 75%. This limit appears to be set by the ratio of the first-order rate constants for interconversion of inactive covalently modified enzyme and a readily dissociable non-covalent enzyme-modifier complex. 2. Reactivation was virtually complete on 150-fold dilution: first-order analysis yielded an estimate of the rate constant (0.164min-1), which was then used in the kinetic analysis of the forward inactivation reaction. This provided estimates for the rate constant for conversion of non-covalent complex into inactive enzyme (0.465 min-1) and the dissociation constant of the non-covalent complex (2.8 mM). From the two first-order constants, the minimum attainable activity in a single cycle of treatment may be calculated as 24.5%, very close to the observed value. 3. Successive cycles of modification followed by reduction with NaBH4 each decreased activity by the same fraction, so that three cycles with 3.6 mM-pyridoxal 5'-phosphate decreased specific activity to about 1% of the original value. The absorption spectrum of the enzyme thus treated indicated incorporation of 2-3 mol of pyridoxal 5'-phosphate per mol of subunit, covalently bonded to lysine residues. 4. NAD+ and NADH protected the enzyme completely against inactivation by pyridoxal 5'-phosphate, but ethanol and acetaldehyde were without effect. 5. Pyridoxal 5'-phosphate used as an inhibitor in steady-state experiments, rather than as an inactivator, was non-competitive with respect to both NADH and acetaldehyde. 6. The partially modified enzyme (74% inactive) showed unaltered apparent Km values for NAD+ and ethanol, indicating that modified enzyme is completely inactive, and that the residual activity is due to enzyme that has not been covalently modified. 7. Activation by methylation with formaldehyde was confirmed, but this treatment does not prevent subsequent inactivation with pyridoxal 5'-phosphate. Presumably different lysine residues are involved. 8. It is likely that the essential lysine residue modified by pyridoxal 5'-phosphate is involved either in binding the coenzymes or in the catalytic step. 9. Less detailed studies of yeast alcohol dehydrogenase suggest that this enzyme also possesses an essential lysine residue.     

Evidence for an essential lysine in glucose-6-phosphate dehydrogenase from Leuconostoc mesenteroides.

1. Pyridoxal 5'-phosphate inhibits glucose-6-phosphate dehydrogenase from Leuconostoc mesenteroides reversibly which Ki equals 0.04-0.06 mM. 2. This inhibition is competitive with respect to glucose 6-phosphate and non-competitive with respect to NADP+ or NAD+. Interaction between enzyme and excess pyridoxal 5'-phosphate follows pseudo-first-order kinetics and indicates that one molecule of inhibitor reacts with each active unit of enzyme. 3. Substrate and coenzyme protect the enzyme from inhibition by pyridoxal 5'-phosphate. Dissociation constants for NADP+ and glucose 6-phosphate were determined from their effects on the kinetics of enzyme--inhibitor interaction. 4. Reaction of the enzyme with pyridoxal 5'-phosphate produces a typical Schiff-base absorbance peak at 430 nm. Subsequent reduction with sodium borohydride leads to spectral changes characteristic for the formation of a secondary amine. 5. The irreversibly inactivated enzyme thus produced contains two moles of inhibitor per mole of enzyme (two subunits per mole). After protein hydrolysis, N-6-pyridoxyllysine can be identified by paper chromatography. 6. The enzyme is inhibited irreversibly by 1-fluoro-2,4-dinitrobenzene, even in the presence of excess 2-mercaptoethanol. At least one dinitrophenyl group is bound per active unit of enzyme; 4 to 5 moles of dinitrophenyl group are bound per mole of enzyme. NADP+ AND GLUCOSE 6-PHOSPHATE PROTECT AGAINST INHIBITION BY 1-FLUORO-2,4-DINITROBENZENE. The absorption spectrum of dinitrophenyl-enzyme corresponds to that for dinitrophenylated amino groups. 7. These studies indicate that there is an essential lysine at the active site of the enzyme. It is suggested that the function of this lysine is to bind glucose 6-phosphate. 8. It is proposed that a group of "active lysine" proteins may exist (in analogy with the "active serine" enzymes), which share a common structural feature at their substrate-binding site and to which pyridoxal 5'-phosphate binds specifically.     

Inhibition by ethylene of polyamine biosynthetic enzymes enhanced lysine decarboxylase activity and cadaverine accumulation in pea seedlings

Exposing etiolated pea seedlings to ethylene which inhibited the activity of arginine decarboxylase and S-adenosylmethionine decarboxylase caused an increase in the level of cadaverine. The elevated level of cadaverine resulted from an increase in lysine decarboxylase activity in the tissue exposed to ethylene. The hormone did not affect the apparent K_m of the enzyme, but the apparent V_max was increased by 96%. While lysine decarboxylase activity in the ethylene-treated plants increased in both the meristematic and the elongation zone tissue, cadaverine accumulation was observed in the latter only. The enhancement by ethylene of the enzyme activity was reversed completely 24 hours after transferring the plants to an ethylene-free atmosphere. It is postulated that the increase in lysine decarboxylase activity, and the consequent accumulation of cadaverine in ethylene-treated plants, is of a compensatory nature as a response to the inhibition of arginine and S-adenosylmethionine decarboxylase activity provoked by ethylene.     

Evaluations of tyrosine apodecarboxylase assays for pyridoxal phosphate

The alternate procedures used in the tyrosine apodecarboxylase assays for pyridoxal 5'-phosphate were evaluated to determine optimal conditions. Two preparations of tyrosine apodecarboxylase from Streptococcus faecalis were used: a cell suspension and a partially purified cell-free form. The activity of the decarboxylase was measured in two different assays using [14C]tyrosine or [3H]tyrosine as substrate. The presence of serum proteins caused greater inhibition of the assay for serum pyridoxal phosphate using [14C]tyrosine as substrate than the assay with [3H]tyrosine. In contrast, addition of deproteinized serum extract did not appear to inhibit either assay. The rate of reconstitution of the apodecarboxylase in the cell suspension was at least four times slower than that of the cell-free enzyme. The rate of reconstitution of the cell-free enzyme was faster in acetate than in citrate buffer. Inorganic sulfate or phosphate, at normal plasma concentrations, did not alter either the reconstitution rate of tyrosine decarboxylase or the final activity obtained in the assays using either substrate. The tyrosine apodecarboxylase assay for pyridoxal phosphate can be optimized by using deproteinized sera or plasma and incubating the cell-free apoenzyme with the coenzyme in acetate buffer for a time sufficient to obtain maximum reconstitution.