Synthesis of Extracellular L-lysine-α-oxidase along with Degrading Enzymes by Trichoderma cf. aureoviride Rifai VKM F-4268D: Role in Biocontrol and Systemic Plant Resistance.

When cultivating on wheat bran or deactivated fungal mycelium as a model of "natural growth", the ability of Trichoderma to synthesize extracellular L-lysine-α-oxidase (LysO) simultaneously with cell-wall-degrading enzymes (proteases, xylanase, glucanases, chitinases, etc.), responsible for mycoparasitism, was shown. LysO, in turn, causes the formation of H2O2 and pipecolic acid. These compounds are known to be signaling molecules and play an important role in the induction and development of systemic acquired resistance in plants. Antagonistic effects of LysO have been demonstrated against phytopathogenic fungi and Gram-positive or Gram-negative bacteria with dose-dependent cell death. The antimicrobial effect of LysO decreased in the presence of catalase. The generating intracellular ROS in the presence of LysO was also shown in both bacteria and fungi, which led to a decrease in viable cells. These results suggest that the antimicrobial activity of LysO is due to two factors: the formation of exogenous hydrogen peroxide as a product of the enzymatic oxidative deamination of L-lysine and the direct interaction of LysO with the cell wall of the micro-organisms. Thus, LysO on its own enhances the potential of the producer in the environment; namely, the enzyme complements the strategy of the fungus in biocontrol and indirectly participates in inducing SAR and regulating the relationship between pathogens and plants.

Fungal Enzyme l-Lysine α-Oxidase Affects the Amino Acid Metabolism in the Brain and Decreases the Polyamine Level.

The fungal glycoprotein l-lysine α-oxidase (LO) catalyzes the oxidative deamination of l-lysine (l-lys). LO may be internalized in the intestine and shows antitumor, antibacterial, and antiviral effects in vivo. The main mechanisms of its effects have been shown to be depletion of the essential amino acid l-lys and action of reactive oxidative species produced by the reaction. Here, we report that LO penetrates into the brain and is retained there for up to 48 h after intravenous injection, which might be explained by specific pharmacokinetics. LO actively intervenes in amino acid metabolism in the brain. The most significant impact of LO was towards amino acids, which are directly exposed to its action (l-lys, l-orn, l-arg). In addition, the enzyme significantly affected the redistribution of amino acids directly associated with the tricarboxylic acid (TCA) cycle (l-asp and l-glu). We discovered that the depletion of l-orn, the precursor of polyamines (PA), led to a significant and long-term decrease in the concentration of polyamines, which are responsible for regulation of many processes including cell proliferation. Thus, LO may be used to reduce levels of l-lys and PA in the brain.

Structural changes in the cell envelope of Yarrowia lipolytica yeast under stress conditions.

Ultrastructural changes in the cell envelope of the yeast Yarrowia lipolytica as a stress response were examined using electron microscopy. The formation of new cellular surface structures, including membrane vesicles, pore channels, and wall surface globules, were shown for the first time under conditions of oxidative (endogenous and exogenous) or thermal stress. This demonstrates once again that under stress conditions the microorganisms reveal properties previously unknown for them. Particularly noteworthy is the accumulation of silicon in the surface globules, which was revealed by X-ray microanalysis of the elemental composition of thin sections of cells. A multilayered plasmalemma instead of a 3-layered one is also characteristic for stressed cells. The envelope modifications above were observed only as a stress response and were not detected in stationary-growth-phase yeast cells that assume different physiological states. A decrease in the intracellular level of cAMP allows us to assume that a common factor activates defensive mechanisms thus explaining the similarity of the response under different stress conditions. The data presented not only enable visualization of the yeast stress response and add to our awareness of the diversity of adaptive reactions, but they also raise questions about the interrelations of the stress phenomena and their functional necessity in the cell.

Kinetic characteristics of L-lysine α- oxidase from Trichoderma cf. aureoviride Rifai VKM F-4268D: Substrate specificity and allosteric effects.

The present work aims to investigate the kinetic characteristics of homodimer enzyme L-lysine α-oxidase from Trichoderma cf. aureoviride Rifai VKM F-4268D, taking into account allosteric effects. The enzyme was first shown to reveal positive cooperativeness, h=2.05±0.15. Using additional opportunities of Hill coefficient the value of the Michaelis-Menten constant has been estimated, Km=1.015∙10-5М, indicating high strength of substrate binding to the active site of each subunit. High selectivity and absolute L-stereospecificity of the enzyme were shown. The inhibition of L-lysine conversion by non-cleavable lysine analogs as well as the reaction product was found out to take place. These effects have been evaluated only as the inhibition coefficients (%). A more detailed study of these inhibition effects was complicated because of the cooperativeness of enzyme subunits mentioned above. The kinetic scheme of L-lysine α-oxidase was proposed involving parallel-subsequent action of each of two subunits in the catalytic act. We think that the results obtained will be useful for studying the kinetic properties of other multi-subunit enzymes and improve understanding of the mechanisms of their action.

Enzymatic properties and anticancer activity of L-lysine α-oxidase from Trichoderma cf. aureoviride Rifai BKMF-4268D.

L-Lysine α-oxidase (LO) from a novel Trichoderma strain: Trichoderma cf. aureoviride Rifai shows favorable biochemical and kinetic properties (Km for L-lysine of 17.9 µmol/l, optimum pH 8.0, high stability) and significant antiproliferative activity both in vitro and in vivo. The molecular weight of LO was determined to be 115-116 kDa; the active dimer consists of two identical 57-58 kDa subunits. LO shows considerable cytotoxicity against the following tumor cell lines: K562, LS174T, HT29, SCOV3, PC3, and MCF7, with the inhibition concentration (IC50) ranging from 3.0×10 to 7.8×10 U/ml (3.2×10 to 8.2×10 mg/ml). Two human colon cancer xenografts HCT116 and LS174T and breast adenocarcinoma T47D implanted subcutaneously into Balb/c nude mice showed high sensitivity to LO with a T/C of 12, 37, and 36%, respectively (P<0.05). The antitumor efficacy of LO was observed in the absence of pronounced morbidity or toxicity in vivo. Taken together, these data suggest that LO may be considered as an effective anticancer agent for the treatment of solid tumors in vivo. This study presents promising data on the possible application of LO in clinical oncology for patients with colorectal cancer.

Gossypol Inhibits Electron Transport and Stimulates ROS Generation in Yarrowia lipolytica Mitochondria.

This work studied the effect of gossypol on the mitochondrial respiratory chain of Yarrowia lipolytica. The compound was shown to inhibit mitochondrial electron transfer and stimulate generation of reactive oxygen species. The inhibition kinetics in oxidation of various substrates (NADH, succinate, α-glycerophosphate and pyruvate + malate) by isolated mitochondria was investigated. Analysis of the kinetic parameters showed gossypol to inhibit two fragments of the mitochondrial electron transfer chain: a) between coenzyme Q and cytochrome b with K(IIIi) of 118.3 µM (inhibition by the noncompetitive type), and b) at the level of exogenous NADH dehydrogenase with of K(Ii) 17.2 µM (inhibition by the mixed type).

Reactivation of the alternative oxidase of Yarrowia lipolytica by nucleoside monophosphates.

The study of the effect of nucleoside phosphates on the activity of cyanide-resistant oxidase in the mitochondria and submitochondrial particles of Yarrowia lipolytica showed that adenosine monophosphate (5'-AMP, AMP) did not stimulate the respiration of intact mitochondria. The incubation of mitochondria at room temperature (25 degrees C) for 3-5 h or their treatment with ultrasound, phospholipase A, and the detergent Triton X-100 at a low temperature inactivated the cyanide-resistant alternative oxidase. The inactivated alternative oxidase could be reactivated with AMP. The reactivating effect of AMP was enhanced by azolectin. Some other nucleoside phosphates also showed reactivating ability in the following descending order: AMP = GMP > GDP > GTP > MP > IMP. The apparent K(m) values for AMP in reactivation of the alternative oxidase of submitochondrial particles or mitochondria treated with Triton X-100 and incubated at 25 degrees C were calculated. Physiological aspects of activation of the alternative oxidase are discussed in connection with the impairment of electron transfer through the cytochrome pathway.

Involvement of the alternative oxidase in respiration of Yarrowia lipolytica mitochondria is controlled by the activity of the cytochrome pathway.

The degree of involvement of cyanide-resistant alternative oxidase in the respiration of Yarrowia lipolytica mitochondria was evaluated by comparing the rate of oxygen consumption in the presence of cyanide, which shows the activity of the cyanide-resistant alternative oxidase, and the oxidation rate of cytochrome c by ferricyanide, which shows the activity of the main cytochrome pathway. The oxidation of succinate by mitochondria in the presence of ferricyanide and cyanide was associated with oxygen consumption due to the functioning of the alternative oxidase. The subsequent addition of ADP or FCCP (an uncoupler of oxidative phosphorylation) completely inhibited oxygen consumption by the mitochondria. Under these conditions, the inhibition of the alternative oxidase by benzohydroxamic acid (BHA) failed to affect the reduction of ferricyanide at the level of cytochrome c. BHA did not influence the rate of ferricyanide reduction by the cytochrome pathway occurring in controlled state 4, nor could it change the phosphorylation quotient ATP/O upon the oxidation of various substrates. These data indicate that the alternative system is unable to compete with the cytochrome respiratory chain for electrons. The alternative oxidase only transfers the electrons that are superfluous for the cytochrome respiratory chain.