Biochemical stability and molecular dynamic characterization of Aspergillus fumigatus cystathionine γ-lyase in response to various reaction effectors |
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| Institution: | 1. Microbiology and Botany Department, Faculty of Science, Zagazig University, Zagazig 44519, Egypt;2. Mid-Florida Research and Education Center, University of Florida/ Institute of Food and Agricultural Sciences, Apopka, FL, USA;3. Kaust Catalysis Center, King Abdullah University of Science and Technology, Thuwal, Saudi Arabia;4. Department of Medical Biochemistry and Molecular Biology, Faculty of Medicine, Zagazig University, Zagazig 44519, Egypt;5. Department of Nanoengineering, University of California, San Diego, UCSD, CA, USA;1. Division of Pediatric Nephrology, Department of Pediatrics, Postgraduate Institute of Medical Education & Research and Associated Dr Ram Manohar Lohia Hospital, Baba Kharak Singh Marg, New Delhi, 110001, India;2. Division of Pediatric Nephrology, Department of Pediatrics, Lady Hardinge Medical College and Associated Kalawati Saran Children Hospital, New Delhi, 110001, India;3. Genomics and Molecular Medicine, CSIR-Institute of Genomics and Integrative Biology, New Delhi, India;4. Academy of Scientific and Innovative Research, Delhi, India;5. Department of Pathology, GB Pant Hospital, New Delhi, India;6. Department of Biostatistics, All India Institute of Medical Sciences, New Delhi, India;1. College of Chemistry and Pharmaceutical Engineering, Nanyang Normal University, 473061 Nanyang, PR China;2. School of Metallurgy and Chemical Engineering, Jiangxi University of Science and Technology, Ganzhou, Jiangxi Province 341000, PR China;1. Department of Biochemistry. Protein Research Chair. College of Science. King Saud University, PO Box 2455, Riyadh 11451, Saudi Arabia;2. Vaccines and Biologics Research Unit. Department of Pharmaceutics. College of Pharmacy. King Saud University, PO Box 2457, Riyadh, 11451, Saudi Arabia;3. Zoology Department. College of Science. King Saud University, Riyadh, Saudi Arabia;3. Department of Pathology, New York Medical College, Valhalla, New York 10595;4. Department of Physiology and Pharmacology, University of Toledo, Toledo, Ohio 43614 |
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| Abstract: | 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 13.8 U/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. |
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| Keywords: | Cystathionine γ-lyase Conformational structure Electrostatics Molecular dynamics MMPBSA Thermal shift assay |
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