Ashraf S. A. El-Sayed

Microbial l-methioninase: production, molecular characterization, and therapeutic applications

Abstractl-Methioninase is ubiquitous in all organisms except in mammals. It mainly catalyzes the, α, γ-elimination of l-methionine to α-ketobutyrate, methanethiol, and ammonia. Unlike normal cells, methionine dependency was reported as a biochemical phenomenon among various types of cancer cells. Thus, l-methioninase is the universal protocol for triggering the majority of tumor cells. This review is an attempt to briefly describe the occurrence of the biochemical and molecular properties of l-methioninase by a comparative manner to the eukaryotic and prokaryotic source for the maximum exploitation in the therapeutic field. The combination of l-methioninase treatment, gene therapy, and chemotherapeutic drugs clearly explores the various therapeutic aspects of this enzyme. Finally, the perspectives for increasing the therapeutic efficacy of this enzyme were described.

Purification and characterization of a new L-methioninase from solid cultures of Aspergillus flavipes

L-Methioninase was purified to electrophoretic homogeneity from cultures of Aspergillus flavipes using anion-exchange and gel filtration chromatography by 12.1 fold compared to the crude enzyme preparation. The purified enzyme had a molecular mass of 47 kDa under denaturing conditions and an isoelectric point of 5.8 with no structural glycosyl residues. The enzyme had optimum activity at pH 7.8 and pH stability from 6.8–8.0 at 35°C. The enzyme appeared to be catalytically stable below 40°C. The enzyme activity was strongly inhibited by DL-propargylglycine, hydroxylamine, PMSF, 2-mercaptoethanol, Hg+, Cu2+, and Fe2+, with slight inhibition by Triton X-100. A flavipes L-methioninase has a higher catalytic affinity towards L-methionine (Km, 6.5 mM and Kcat, 14.1 S−1) followed by a relative demethiolating activity to L-homo-cysteine (Km, 12 mM and Kcat, 9.3 S−1). The enzyme has two absorption maxima at 280 and 420 nm, typical of other PLP-enzymes. Apo-L-methioninase has the ability to reconstitute its structural catalytic state completely upon addition of 0.15 mM PLP. L-Methioninase has neither an appreciable effect on liver function, platelet aggregation, nor hemolysis of human blood. The purified L-methioninase from solid cultures of A. flavipes displayed unique biochemical and catalytic properties over the currently applied Pseudomonad enzyme.

L-glutaminase production by Trichoderma koningii under solid-state fermentation

Solid state fermentation was conducted for the production of L-glutaminase by Trichoderma koningii Oud.aggr. using different agro-industrial byproducts inlcuding wheat bran, groundnut residues, rice hulls, soya bean meal, corn steep, sesamum oil cake, cotton seed residues and lentil industrial residues as solid substrates. Wheat bran was the best substrate for induction of L-glutaminase (12.1 U/mg protein) by T. koningii. The maximum productivity (23.2 U/mg protein) and yield (45.0 U/gds) of L-glutaminase by T. koningii occurred using wheat bran of 70% initial moisture content, initial pH 7.0, supplemented with D-glucose (1.0%) and L-glutamine (2.0% w/v), inoculated with 3 ml of 6 day old fungal culture and incubated at 30°C for 7 days. After optimization, the productivity of L-glutaminase by the solid cultures of T. koningii was increased by 2.2 fold regarding to the submerged culture.

L-methioninase production by Aspergillus flavipes under solid-state fermentation

Solid‐state fermentation was carried out for the production of extra‐cellular L‐methioninase by Aspergillus flavipes (Bain and Sart.) using nine agro‐industrial residues, namely wheat bran, rice bran, wheat flour, coconut seeds, cotton seeds, ground nut cake, lentil hulls, soya beans and chicken feathers. Chicken feathers were selected as solid substrate for L‐methioninase production by A. flavipes. The maximum L‐methioninase productivity (71.0 U/mg protein) and growth (11 mg protein/ml) of A. flavipes was obtained using alkali pretreated chicken feathers of 50% initial moisture content as substrate supplemented with D‐glucose (1.0% w/v) and L‐methionine (0.2% w/v). External supplementation of the fermentation medium with various vitamin sources has no overinductive effect on L‐methioninase biosynthesis. The partially purified A. flavipes L‐methioninase preparation showed highest activity (181 U/ml) at pH 8.0 with stability over a pH range (pH 6–8) for 2 h. L‐methioninase activity was increased by preincubation of the enzyme for 2 h with Co2+, Mn2+, Cu2+ and Mg2+ and strongly inhibited by the presence of EDTA, NaN3, Li2+, Cd2+, DMSO and 2‐mercaptoethanol. The enzyme preparation has a broad substrate spectrum showing a higher affinity to deaminate L‐glycine, N ‐acetylglucosamine and glutamic acid, in addition to their proteolytic activity against bovine serum albumin, casein, gelatin and keratin. The partially purified enzyme was found to be glyco‐metalloproteinic in nature as concluded from the analytical and spectroscopic profiles of the enzyme preparation. The demethiolating activity of the enzyme was also visualized chromogenially. (© 2009 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)

Co-immobilization of PEGylated Aspergillus flavipes L-methioninase with glutamate dehydrogenase: a novel catalytically stable anticancer consortium.

Aspergillus flavipes L-methioninase (AfMETase) exhibits reliable pharmacokinetic properties and anticancer potency in vitro[10]. To maximize its therapeutic efficiency as protection against in vivo proteolysis, reduction of antigenicity and hyperammoniemia, the enzyme was PEGylated and coupled with glutamate dehydrogenase (GDH). The highest degree of PEGylation was measured at 40-50/1 molar ratio of PEG to AfMETase, with a lower mobility on SDS-PGE, compared to the native AfMETase. The activity of free AfMETase was reduced to 66.2% and further to 50% upon PEGylation and GDH conjugation, respectively. The highest degree of surface NH2 modification of AfMETase-GDH co-immobilizates (65%), was reported using 300 mM glutaraldehyde, with 31% methionine conversion. Using L-cysteine and L-methionine as active site protectors, the activity of PEG-AfMETase and PEG-AfMETase-GDH was increased by 14.4 and 32.9-fold, respectively. At 45°C, PEG-AfMETase, PEG-AfMETase-GDH and AfMETase-GDH conjugate have a T1/2 10.3, 8.5 and 7.6 h, inactivation rate (Kr) 0.021, 0.03 and 0.016 min, with 2.0, 1.65 and 1.47-fold stabilization, respectively. Kinetically, the three immobilizates have a relatively similar Km values for L-methionine (7.4-7.9 mM), with lower affinity to homocysteine and cysteine, with stability to PLP-enzyme inhibitors (propargylglycine and hydroxylamine), indicating the protective effect by PEG moieties on the enzyme structure. Also, the three immobilizates exhibited improved stability against proteolysis in vitro, comparing to free AfMETase.

Pharmacokinetics, immunogenicity and anticancer efficiency of Aspergillus flavipes l-methioninase

Methionine starvation can powerfully modulate DNA methylation, cell cycle transition, polyamines and antioxidant synthesis of tumor cells, in contrary to normal ones. Aspergillus flavipesl-methioninase was previously characterized by our studies, displaying affordable biochemical properties comparing to Pseudomonas putida enzyme (ONCASE). Thus, the objective of current study was to evaluate the catalytic properties of Af-METase in New Zealand rabbits, exploring its antitumor efficacy. In vivo, Af-METase (40.8 U/ml) have T(1/2) 19.8 h, elimination constant 0.088 U/h and apparent volume distribution 85 U/ml. Also, Af-METase has two maxima one at A(280 nm) (apo-enzyme) and at A(420 nm) (internal Schiff base of PLP), unlike control plasma (without enzyme). The two peaks of absorption spectra were detected maximally at 15 min then the absorbance at 420 nm was subsequently decreased with circulation time, due to dissociation of the co-enzyme. The A₂₈₀/₄₂₀ ratio was increased from 1.69 to 5.81 with circulation time from 15 to 30 h. Rabbits plasma methionine was depleted from 18.7 μM (control) to 8.8 μM after 1h of enzyme injection and completely omitted after 2 h till 19 h, assuming the sustainability of negligible levels of methionine (< 2 μM) in plasma of rabbits, for about 17 h. Upon infusion of PLP, the T(½) of Af-METase was significantly prolonged by 3.2 fold, assuming the fully reconstitution of the enzyme. The holo-AfMETase still retained its co-enzyme, completely, till 33 h of PLP infusion. From spectral studies, the internal aldimine linkage of apo-Af-METase was constructed upon PLP infusion, with fully catalytic structure after less than 4h of its infusion, the A₂₈₀/₄₂₀ ratio being not relatively changed till 45 h. After 25 days of last enzyme dose, the titer of IgG was increase by about 1.66 fold comparing to control (without enzyme). However, IgM was not detected along the tested challenge points. In vitro, plasma anti-Af-METase neutralizing antibodies (NAb) were assessed, with no significant reduction on activity of Af-METase by Nab. All the hematological parameters were in normal range, otherwise, the RBCs titer and platelet level was slightly increased, after 25 days of Af-METase injection, comparing to control. There is no obvious negative effect on chemistry of liver, kidney, glucose, lipids, and other electrolytes. Additionally, the anticancer activity of Af-METase was evaluated against five types of human cancer cell lines, in vitro. The enzyme showed a powerful activity against prostate (PC3), liver (HEPG2) and breast (MCF7) cancers, with IC₅₀ 0.001 U/ml, 0.26 U/ml and 0.37 U/ml, respectively.

Purification, immobilization, and biochemical characterization of l‐arginine deiminase from thermophilic Aspergillus fumigatus KJ434941: Anticancer activity in vitro

l‐Arginine deiminase (ADI) has a powerful anticancer activity against various tumors, via arginine depletion, arresting the cell cycle at G1 phase. However, the current clinically tried bacterial ADI displayed a higher antigenicity and lower thermal stability. Thus, our objective was to purify and characterize this enzyme from thermophilic fungi, to explore its catalytic and antigenic properties for therapeutic uses. ADI was purified from thermophilic Aspergillus fumigatus KJ434941 to its electrophoretic homogeneity by 5.1‐fold, with molecular subunit 50 kDa. The purified ADI was PEGylated and covalently immobilized on dextran to explore its catalytic properties. The specific activity of free ADI, PEG‐ADI, and Dex‐ADI was 26.7, 21.5, and 18.0 U/mg, respectively. At 50°C, PEG‐ADI displays twofold resistance to thermal denaturation (t1/2 13.9 h), than free ADI (t1/2 6.9 h), while at 70°C, the thermal stability of PEG‐ADI was increased by 1.7‐fold, with similar stability to Dex‐ADI with the free one. Kinetically, free ADI had the higher catalytic affinity to arginine, followed by PEG‐ADI and Dex‐ADI. Upon proteolysis for 30 min, the residual activity of native ADI, PEG‐ADI, and Dex‐AD was 8.0, 32.0, and 20.0% for proteinase K and 10.0, 52.0, and 90.0% for acid protease, respectively. The anticancer activity of the ADIs was assessed against HCT, HEP‐G2, and MCF7, in vitro. The free and PEG‐ADI exhibits a similar cytotoxic efficacy for the tested cells, lower than Dex‐ADI. The free ADI had IC50 value 22.0, 16.6, and 13.9 U/mL, while Dex‐ADI had 3.98, 5.18, and 4.43 U/mL for HCT, MCF7, and HEPG‐2, respectively. The in vitro anticancer activity of ADI against HCT, MCF7, and HEPG‐2 was increased by five‐, three‐, and threefold upon covalent modification by dextran. The biochemical and hematological parameters of the experimented animals were not affected by ADIs dosing, with no signs of anti‐ADI immunoglobulins in vivo. The in vivo half‐life time of free ADI, PEG‐ADI, and Dex‐ADI was 29.7, 91.1, 59.6 h, respectively. The present findings explored a novel thermostable, less antigenic ADI from thermophilic A. fumigatus, with further molecular and crystallographic analyses, this enzyme will be a powerful candidate for clinical trials.

Molecular cloning, biochemical characterization, and antitumor properties of a novel L-asparaginase from Synechococcus elongatus PCC6803

L-asparaginase (EC 3.5.1.1), which catalyzes the deamidation of L-asparagine to L-aspartic acid and ammonia, has been widely used as a key therapeutic tool in the treatment of tumors. The current commercially available L-asparaginases, produced from bacteria, have signs of toxicity and hypersensitivity reactions during the course of tumor therapy. Therefore, searching for L-asparaginases with unique biochemical properties and fewer adverse effects was the objective of this work. In this study, cyanobacterial strain Synechococcus elongatus PCC6803 was found as a novel source of L-asparaginase. The L-asparaginase gene coding sequence (gi:939195038) was cloned and expressed in E. coli BL21(DE3), and the recombinant protein (Se.ASPII) was purified by affinity chromatography. The enzyme has high affinity towards Lasparagine and shows very weak affinity towards L-glutamine. The enzymatic properties of the recombinant enzyme were investigated, and the kinetic parameters (Km, Vmax) were measured. The pH and temperature dependence profiles of the novel enzyme were analyzed. The work was extended to measure the antitumor properties of the novel enzyme against different human tumor cell lines.

Genome editing approaches: manipulating of lovastatin and taxol synthesis of filamentous fungi by CRISPR/Cas9 system

Filamentous fungi are prolific repertoire of structurally diverse secondary metabolites of remarkable biological activities such as lovastatin and paclitaxel that have been approved by FDA as drugs for hypercholesterolemia and cancer treatment. The clusters of genes encoding lovastatin and paclitaxel are cryptic at standard laboratory cultural conditions (Kennedy et al. Science 284:1368–1372, 1999; Bergmann et al. Nature Chem Biol 3:213–217, 2007). The expression of these genes might be triggered in response to nutritional and physical conditions; nevertheless, the overall yield of these metabolites does not match the global need. Consequently, overexpression of the downstream limiting enzymes and/or blocking the competing metabolic pathways of these metabolites could be the most successful technologies to enhance their yield. This is the first review summarizing the different strategies implemented for fungal genome editing, molecular regulatory mechanisms, and prospective of clustered regulatory interspaced short palindromic repeat/Cas9 system in metabolic engineering of fungi to improve their yield of lovastatin and taxol to industrial scale. Thus, elucidating the putative metabolic pathways in fungi for overproduction of lovastatin and taxol was the ultimate objective of this review.

Biochemical stability and molecular dynamic characterization of Aspergillus fumigatus cystathionine γ-lyase in response to various reaction effectors.

Cystathionine γ-lyase (CGL) is a key enzyme in the methionine-cysteine cycle in all living organisms forming cysteine, α-ketobutyrate and ammonia via homocysteine and cystathionine intermediates. Although, human and plant CGLs have been extensively studied at the molecular and mechanistic levels, there has been little work on the molecular and catalytic properties of fungal CGL. Herein, we studied in detail for the first time the molecular and catalytic stability of Aspergillus fumigatus CGL, since conformational instability, inactivation and structural antigenicity are the main limitations of the PLP-dependent enzymes on various therapeutic uses. We examined these properties in response to buffer compositions, stabilizing and destabilizing agents using Differential Scanning Fluorometery (DSF), steady state and gel-based fluorescence of the intrinsic hydrophobic core, stability of internal aldimine linkage and catalytic properties. The activity of the recombinant A. fumigatus CGL was 13.8U/mg. The melting temperature (Tm) of CGL in potassium phosphate buffer (pH 7.0-8.0) was 73.3°C, with ∼3°C upshifting in MES and sodium phosphate buffers (pH 7.0). The conformational thermal stability was increased in potassium phosphate, sodium phosphate and MES buffers, in contrast to Tris-HCl, HEPES (pH 7.0) and CAPS (pH 9.0-10.0). The thermal stability and activity of CGL was slightly increased in the presence of trehalose and glycerol that might be due to hydration of the enzyme backbone, unlike the denaturing effect of GdmCl and urea. Modification of surface CGL glutamic and aspartic acids had no significant effect on the enzyme conformational and catalytic stability. Molecular modeling and dynamics simulations unveil the high conformational stability of the overall scaffold of CGL with high flexibility at the non-structural regions. CGL structure has eight buried Trp residues, which are reoriented to the enzyme surface and get exposed to the solvent under perturbation of destabilizers. Furthermore, electrostatic calculations of selected snapshots of CGL 3D structure under different experimental conditions showed a remarkable differences on the polarity of the enzyme surface.