Evaluation of antileukemic activity of L-lysine-alpha oxidase from Trichoderma sp. in chemotherapeutic experiments

Evidences on the antileukemic effect of L-lysine alpha-oxidase from Trichoderma sp. are presented. It is inferred that enzyme needs detail studying as a potential chemotherapeutic drug both in experimental and clinical research.     

A new antitumor enzyme, L-lysine alpha-oxidase from Trichoderma viride. Purification and enzymological properties

L-Lysine alpha-oxidase from Trichoderma viride Y244-2 has been purified to homogeneity. The enzyme shows absorption maxima at 277, 388, and 466 nm and a shoulder around 490 nm and contains 2 mol of FAD/mol of enzyme. The enzyme has a molecular weight of approximately 116,000 and consists of two subunits identical in molecular weight (about 56,000). In addition to L-lysine, L-ornithine, L-phenylalanine, L-tyrosine, L-arginine, and L-histidine are oxidized by the enzyme to a lesser extent. Several lysine analogs such as delta-hydroxylysine are oxidized efficiently. Balance studies showed that 1 mol of L-lysine is converted to an equimolar amount of alpha-keto-epsilon-aminocaproate, ammonia, and hydrogen peroxide with the consumption of 1 mol of oxygen. alpha-Keto-epsilon-aminocaproate spontaneously is dehydrated intramolecularly into delta 1-piperideine-2-carboxylate in the presence of catalase, and is oxidatively decarboxylated into delta-aminovalerate in the absence of catalase. The Michaelis constants are as follows: 0.04 mM for L-lysine, 0.44 mM for L-ornithine, 14 mM for L-phenylalanine, and 1.6 mM for oxygen with L-lysine.     

Evaluation of intrinsic immobilized kinetics in hollow fiber reactor systems.

Immobilized cell and enzyme hollow fiber reactors have been developed for a variety of biochemical and biomedical applications. Reported mathematical models for predicting substrate conversion in these reactors have been limited in accuracy because of the use of free-solution kinetic parameters. This paper describes a method for determining the intrinsic kinetics of enzymes immobilized in hollow fiber reactor systems using a mathematical model for diffusion and reaction in porous media and an optimization procedure to fit intrinsic kinetic parameters to experimental data. Two enzymes, a thermophilic beta-galactosidase that exhibits product inhibition and L-lysine alpha-oxidase, were used in the analysis. The intrinsic kinetic parameters show that immobilization enhanced the activity of the beta-galactosidase while decreasing the activity of L-lysine alpha-oxidase. Both immobilized enzymes had higher Km values than did the soluble enzyme, indicating less affinity for the substrate. These results are used to illustrate the significant improvement in the ability to predict substrate conversion in hollow fiber reactors.     

Enzyme electrode for specific determination of L-lysine

L-Lysine alpha-oxidase from Trichoderma viride Y244-2 is immobilized in a gelatin support and fixed on a pO(2) sensor. The enzyme electrode obtained is used in a continuous flow system in order to measure the concentration of L-lysine in a fermentor. The sample oxygen-content dependance of the signal is minimized because of the enzyme support properties. The enzyme electrode response is set for lysine concentration from 0.2mM to 4mM. The specificity of lysine is tested with other amino acids. The enzyme membrane for lysine electrode can be used 3000 times or stored six months with good stability.     

Enzymatic synthesis of L-pipecolic acid by Delta1-piperideine-2-carboxylate reductase from Pseudomonas putida

L-Pipecolic acid is a chiral pharmaceutical intermediate. An enzymatic system for the synthesis of L-pipecolic acid from L-lysine by commercial L-lysine alpha-oxidase from Trichoderma viride and an extract of recombinant Escherichia coli cells coexpressing Delta1-piperideine-2-carboxylate reductase from Pseudomonas putida and glucose dehydrogenase from Bacillus subtilis is described. A laboratory-scale process provided 27 g/l of L-pipecolic acid in 99.7% e.e.     

The effect of L-lysine alpha-oxidase from Trichoderma cf. aureoviride Rifai VKM F-4268D on the rat pheochromocytoma PC12 cell line

L-Amino acid oxidases (L-AAO; EC 1.4.3.2) comprise a group of flavoproteins that catalyze oxidative deamination of L-alpha amino acids to corresponding alpha-keto acids, NH₃ and H₂O₂. Most of these enzymes are homodimers with molecular mass of 100–150 kDa that exhibit antiviral, antifungal, antibacterial, and anticancer activity. Among this group of enzymes L-lysine alpha-oxidase (LO) is especially important as its biological effects may differ from the effects of other L-AAO, because this enzyme preferentially oxidizes L-lysine, the essential amino acid for the human body, without any practical effect on other amino acids. Since molecular mechanisms of the cytotoxic action of LO still require better understanding, in this study we have investigated a possible mechanism of action of LO from Trichoderma cf. aureoviride Rifai VKMF-4268D. A rat pheochromocytoma PC12 cell culture was used as a model. Using flow cytometry a dose-dependent cell death induced by LO was shown. The increase in intracellular reactive oxygen species detected by the 2,7-dichlorodihydrofluorescein assay suggests that the oxidative pathway is one of mechanisms underlying the cytotoxic LO action; however, this does not rule out the involvement of other (previously demonstrated) mechanisms of LO effects on cell death.     

The use of a single-fiber reactor for the enzymatic removal of amino acids from solutions

In this article we describe the use of bench-scale single-fiber dialyzers for the development and testing of an immobilized enzyme reactor for the treatment of leukemia. The treatment is based on the enzymatic removal of specific amino acids from the blood of leukemia patients. L-Lysine alpha-oxidase and catalase were coimmobilized within the void space of the porous region of asymmetric hollow-fiber membranes for the removal of L-lysine from simulated human plasma solutions. Hollow-fiber reactor performance was evaluated using a small single-fiber dialyzer (SFD) consisting of a single fiber encased in a protective glass shell. This small reactor affords ease of use, requires small amounts of chemicals and biochemicals, and gives useful reactor performance data. Single-fiber dialyzers were constructed using polyamide fibers with a molecular weight cutoff of 10,000 (PA10 fibers); these fibers demonstrated the best compatibility with and retention of the enzymes. The SFD performance in removing L-lysine from solution was evaluated under both steady and pulsatile flow operation. Pulsatile flow was tested for two reasons: (1) to enhance the radial mass transfer of lysine within the SFD and (2) to simulate the pulsatile flow of blood in dialysis treatment. The use of pulsatile flow increased lysine conversion by 15% over the steady-flow case. Approximately 40% of the lysine was removed from simulated plasma by the SFD in a 4-h experiment using pulsatile flow in the recycle mode.     

Properties of L-lysine epsilon-dehydrogenase from Agrobacterium tumefaciens

Lysine epsilon-dehydrogenase, which has been purified to homogeneity from the extract of Agrobacterium tumefaciens ICR 1600, had a molecular weight of approximately 78,000 and consisted of two subunits identical in molecular weight (about 39,000). The enzyme showed a high substrate specificity. In addition to L-lysine, S-(beta-aminoethyl)-L-cysteine was deaminated by the enzyme, but to a far lesser extent. NAD+ and some NAD+ analogs (deamino-NAD+ and 3-acetylpyridine-NAD+) served as a cofactor. The pH optimum was at about 9.7 for the deamination of L-lysine. Although the NAD+ saturation curve was hyperbolic, a sigmoid saturation curve for L-lysine was obtained with the diluted enzyme solution, in which the dimeric enzyme was predominant. The reversible association of the enzyme to the tetramer was induced either by increasing the enzyme concentration or by addition of L-lysine. The preincubation of the enzyme with 5 mM L-lysine resulted in a 2-fold increase in the activity and gave a hyperbolic saturation curve for L-lysine. Upon modification of SH groups of the enzyme with DTNB, neither the interconversion between the dimer and the tetramer nor the activation by L-lysine occurred. These results indicated that the dimeric enzyme was activated by L-lysine and the activation resulted from the association of two dimeric enzymes to form a tetramer.     

Activation of L-lysine epsilon-dehydrogenase from Agrobacterium tumefaciens by several amino acids and monocarboxylates

The activation of lysine epsilon-dehydrogenase [EC 1.4.1.] by L-lysine was dependent on lysine concentration and was accompanied by association of the dimeric enzymes to a tetramer. The lysine concentration required for the half-maximal activation was 0.28 mM, which was lower than the Km value for L-lysine. In addition to L-lysine, several compounds, which were neither substrates nor inhibitors, activated the enzyme. The compounds which activated the enzyme have common structural characteristics: they have both a carboxyl group and a hydrophobic side chain. These activators also induced the association of the enzyme. The activation of the enzyme occurred well over the pH range 5.0 to 7.5, and the maximal activation was obtained by preincubation for 5 min at 30 degrees C and pH 7.4, when 5 mM L-lysine or 6-aminocaproate was used as an activator. NADH binding experiments indicated that about 2 mol of NADH bind to 1 mol of the tetrameric enzyme: the dimeric enzyme has one catalytic site. Binding experiments with n-[1-14C]heptanoate and L-[U-14C]lysine showed that approximately 2 mol of ligands bind to 1 mol of the dimeric enzyme and L-lysine could not bind to the catalytic site of the enzyme in the absence of NAD+. These results indicate the presence of one catalytic site and two activator binding binding sites in the dimeric enzyme.     

Occurrence of a novel yeast enzyme, L-lysine epsilon-dehydrogenase, which catalyses the first step of lysine catabolism in Candida albicans

The yeast Candida albicans is able to utilize L-lysine as the sole nitrogen and carbon source accompanied by intracellular accumulation of alpha-aminoadipate-delta-semialdehyde. A novel yeast amino acid dehydrogenase catalysing the oxidative deamination of the epsilon-group of L-lysine was found in this yeast. The enzyme, L-lysine epsilon-dehydrogenase, is strongly induced in cells grown on L-lysine as the sole nitrogen source. The enzyme is specific for both L-lysine and NADP+. The Km values were determined to be 0.87 mM for L-lysine and 0.071 mM for NADP+. An apparent Mr of 87,000 was estimated by gel filtration. The enzyme has maximum activity at pH 9.5 and a temperature optimum of 32 degrees C under our assay conditions.     

Spectrophotometric assays for L-lysine alpha-oxidase and gamma-glutamylamine cyclotransferase

A new assay for l-lysine alpha-oxidase is described. In this assay, the oxidized product generated from l-lysine is reacted with semicarbazide to form alpha-keto-epsilon-aminocaproate semicarbazone. Formation of the alpha-keto acid semicarbazone is continuously monitored spectrophotometrically at 248 nm (epsilon 10,160 +/- 240 M(-1) cm(-1)). The method was adapted to provide a new assay for gamma-glutamylamine cyclotransferase. This enzyme catalyzes the conversion of many l-gamma-glutamylamines to 5-oxo-l-proline and free amine. A biologically important substrate is N(epsilon)-(gamma-l-glutamyl)-l-lysine, which is converted to 5-oxo-l-proline and l-lysine by the action of gamma-glutamylamine cyclotransferase. The l-lysine generated from N(epsilon)-(gamma-l-glutamyl)-l-lysine in an endpoint assay is converted to alpha-keto epsilon-aminocaproate semicarbazone in the presence of semicarbazide, excess l-lysine alpha-oxidase, and catalase. The methods were applied to the determination of gamma-glutamylamine cyclotransferase activity of partially purified preparations of the bovine kidney enzyme and to detect gamma-glutamylamine cyclotransferase activity in rat kidney and liver homogenates.     

Plasma pharmacokinetics and tissue distribution of L-lysine α-oxidase from Trichoderma cf. aureoviride RIFAI VKM F- 4268D in mice

L-lysine α-oxidase (LO) is an L-amino acid oxidase with antitumor, antimicrobial and antiviral properties. Pharmacokinetic (PK) studies were carried out by measuring LO concentration in plasma and tissue samples by enzyme immunoassay. L-lysine concentration in samples was measured spectrophotometrically using LO. After single i.v. injection of 1.0, 1.5, 3.0 mg/kg the circulating T_1/2 of enzyme in mice varied from 51 to 74 min and the AUC_0–inf values were 6.54 ± 0.46, 8.66 ± 0.59, 9.47 ± 1.45 μg/ml × h, respectively. LO was distributed in tissues and determined within 48 h after administration with maximal accumulation in liver and heart tissues. Mean time to reach the maximum concentration was highest for the liver—9 h, kidney—1 h and 15 min for the tissues of heart, spleen and brain. T_1/2 of LO in tissues ranged from 7.75 ± 0.73 to 26.10 ± 2.60 h. In mice, plasma L-lysine decreased by 79% 15 min after LO administration in dose 1.6 mg/kg. The serum L-lysine levels remained very low from 1 to 9 h (< 25 μM, 17%), indicating an acute lack of L-lysine in animals for at least 9 h. Concentration of L-lysine in serum restored only 24 h after LO administration. The results of LO PK study show that it might be considered as a promising enzyme for further investigation as a potential anticancer agent.

     

The effect of carboxylates and halides on L-lysine 6-aminotransferase-catalyzed reactions

L-Lysine:2-oxoglutarate 6-aminotransferase catalyzes very slow transamination between L-alanine and 2-oxoglutarate. A high concentration of anions such as formate, acetate and halides greatly accelerated this transamination without affecting the affinity of the enzyme for L-alanine. In contrast, the anions strongly inhibited the normal L-lysine 6-transamination in a competitive manner with L-lysine and in a non-competitive manner with 2-oxoglutarate. This result suggests that the enzyme has an anion binding site which normally binds the carboxyl group of L-lysine. The binding of halides or carboxylates to this site probably induces a conformational change of the enzyme, and results in the inhibition of L-lysine 6-transamination, and in the stimulation of L-alanine transamination. Treatment of the enzyme with an arginine-specific dicarbonyl reagent, phenylglyoxal, led to a loss of the enzyme activity for L-lysine. The activity for L-alanine was not affected, but the stimulating effect of anions on L-alanine transamination was impaired. Thus, it is suggested that an arginine residue(s) plays an important role in the anion binding site.     

A novel gnd mutation leading to increased L-lysine production in Corynebacterium glutamicum

Toward more efficient L-lysine production, we have been challenging genome-based strain breeding by the approach of assembling only relevant mutations in a single wild-type background. Following the creation of a new L-lysine producer Corynebacterium glutamicum AHP-3 that carried three useful mutations (lysC311, hom59, and pyc458) on the relevant downstream pathways, we shifted our target to the pentose phosphate pathway. Comparative genomic analysis for the pathway between a classically derived L-lysine producer and its parental wild-type identified several mutations. Among these mutations, a Ser-361-->Phe mutation in the 6-phosphogluconate dehydrogenase gene (gnd) was defined as a useful mutation for L-lysine production. Introduction of the gnd mutation into strain AHP-3 by allelic replacement led to approximately 15% increased L-lysine production. Enzymatic analysis revealed that the mutant enzyme was less sensitive than the wild-type enzyme to allosteric inhibition by intracellular metabolites, such as fructose 1,6-bisphosphate, D-glyceraldehyde 3-phosphate, phosphoribosyl pyrophosphate, ATP, and NADPH, which were known to inhibit this enzyme. Isotope-based metabolic flux analysis demonstrated that the gnd mutation resulted in 8% increased carbon flux through the pentose phosphate pathway during L-lysine production. These results indicate that the gnd mutation is responsible for diminished allosteric regulation and contributes to redirection of more carbon to the pentose phosphate pathway that was identified as the primary source for NADPH essential for L-lysine biosynthesis, thereby leading to improved product formation.     

Screen-printed enzyme sensors for l-lysine determination

Sensors for the determination of L-lysine in samples of fermentation broth have been developed. Low-cost screen-printed sensors comprising a platinum working electrode, an Ag/AgCl pseudo reference and a carbon counter electrode were used as transducers for the enzyme sensors. L-lysine-(alpha)-oxidase from Trichoderma viride has been immobilized by entrapment into a polyurethane hydrogel. Sensors were characterized for L-lysine with respect to pH value, linear range, reproducibility, repeatability, storage and working stability. The sensitivities to other amino acids were also determined. A batch system with two working electrodes, one with immobilized enzyme and one without was adapted for the determination of L-lysine by differential measurements. Good agreement was found between L-lysine concentrations measured by the enzyme sensors and by a conventional amino acid analyzer.     

Highly stable L-lysine 6-dehydrogenase from the thermophile Geobacillus stearothermophilus isolated from a Japanese hot spring: characterization, gene cloning and sequencing, and expression

L-Lysine dehydrogenase, which catalyzes the oxidative deamination of L-lysine in the presence of NAD, was found in the thermophilic bacterium Geobacillus stearothermophilus UTB 1103 and then purified about 3,040-fold from a crude extract of the organism by using four successive column chromatography steps. This is the first report showing the presence of a thermophilic NAD-dependent lysine dehydrogenase. The product of the enzyme catalytic activity was determined to be Delta1-piperideine-6-carboxylate, indicating that the enzyme is L-lysine 6-dehydrogenase (LysDH) (EC 1.4.1.18). The molecular mass of the purified protein was about 260 kDa, and the molecule was determined to be a homohexamer with subunit molecular mass of about 43 kDa. The optimum pH and temperature for the catalytic activity of the enzyme were about 10.1 and 70 degrees C, respectively. No activity was lost at temperatures up to 65 degrees C in the presence of 5 mM L-lysine. The enzyme was relatively selective for L-lysine as the electron donor, and either NAD or NADP could serve as the electron acceptor (NADP exhibited about 22% of the activity of NAD). The Km values for L-lysine, NAD, and NADP at 50 degrees C and pH 10.0 were 0.73, 0.088, and 0.48 mM, respectively. When the gene encoding this LysDH was cloned and overexpressed in Escherichia coli, a crude extract of the recombinant cells had about 800-fold-higher enzyme activity than the extract of G. stearothermophilus. The nucleotide sequence of the LysDH gene encoded a peptide containing 385 amino acids with a calculated molecular mass of 42,239 Da.     

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.     

Lysyl-tRNA synthetase from Bacillus stearothermophilus: the Trp314 residue is shielded in a non-polar environment and is responsible for the fluorescence changes observed in the amino acid activation reaction

Three Trp variants of lysyl-tRNA synthetase from Bacillus stearothermophilus, in which either one or both of the two Trp residues within the enzyme (Trp314 and Trp332) were substituted by a Phe residue, were produced by site-directed mutagenesis without appreciable loss of catalytic activity. The following two phenomena were observed with W332F and with the wild-type enzyme, but not with W314F: (1) the addition of L-lysine alone decreased the protein fluorescence of the enzyme, but the addition of ATP alone did not; (2) the subsequent addition of ATP after the addition of excess L-lysine restored the fluorescence to its original level. Fluorometry under various conditions and UV-absorption spectroscopy revealed that Trp314, which was about 20A away from the lysine binding site and was shielded in a non-polar environment, was solely responsible for the fluorescence changes of the enzyme in the L-lysine activation reaction. Furthermore, the microenvironmental conditions around the residue were made more polar upon the binding of L-lysine, though its contact with the solvent was still restricted. It was suggested that Trp314 was located in a less polar environment than was Trp332, after comparison of the wavelengths at the peaks of fluorescence emission and of the relative fluorescence quantum yields. Trp332 was thought, based on the fluorescence quenching by some perturbants and the chemical modification with N-bromosuccinimide, to be on the surface of the enzyme, whereas Trp314 was buried inside. The UV absorption difference spectra induced by the L-lysine binding indicated that the state of Trp314, including its electrostatic environment, changed during the process, but Trp332 did not change. The increased fluorescence from Trp314 at acidic pH compared with that at neutral pH suggests that carboxylate(s) are in close proximity to the Trp314 residue.     

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.     

Effect of gluconic acid as a secondary carbon source on non-growing L-lysine producers cells of Corynebacterium glutamicum. Purification and properties of 6-phosphogluconate dehydrogenase

We studied the production of L-lysine in Corynebacterium glutamicum ATCC 21543 non growing cells obtained by nutrient limitation. Statistical analysis revealed significant differences in the L-lysine titers of glucose, gluconic acid or glucose-gluconic acid cultures. Higher L-lysine titer obtained in batch cultures with mixed carbon sources or gluconic acid alone were found to be associated with a high 6-phosphogluconate dehydrogenase activity (6PGDH, E.C.1.1.1.44). This enzyme is a pivotal enzyme within the hexose monophosphate pathway, and thus of importance for L-lysine production. 6PGDH was purified and characterized. The purified enzyme migrates as a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis with a molecular mass of 52.5 kDa. The molecular mass of the native enzyme was estimated to be 120 kDa by molecular exclusion chromatography, thus suggesting a homodimeric structure. The amino terminal sequence shows a strong similarity (a match of 86% of the first 20 amino acid) to the 6PGDH from other microorganisms such as, E. coli and B. subtilis. The pI of the dimeric native enzyme and the optimum pH were 6.2 and 8.0, respectively. For the oxidative decarboxylation of 6-phosphogluconate, K_m of 71 μM and 43 μM were obtained for 6-phosphogluconate and NADP⁺, respectively.