title: Biochemical stability and molecular dynamic characterization of Aspergillus fumigatus cystathionine γ-lyase in response to various reaction effectors.
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 13.8U/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.
authors.affiliation: Microbiology and Botany Department, Faculty of Science, Zagazig University, Zagazig 44519, Egypt; Mid-Florida Research and Education Center, University of Florida/ Institute of Food and Agricultural Sciences, Apopka, FL, USA. Electronic address: ashrafzu@ufl.edu.
authors.forename: Ashraf S A
authors.initials: AS
authors.lastname: El-Sayed
authors.affiliation: Kaust Catalysis Center, King Abdullah University of Science and Technology, Thuwal, Saudi Arabia.
authors.forename: Safwat
authors.initials: S
authors.lastname: Abdel-Azeim
authors.affiliation: Department of Medical Biochemistry and Molecular Biology, Faculty of Medicine, Zagazig University, Zagazig 44519, Egypt.
authors.forename: Hend M
authors.initials: HM
authors.lastname: Ibrahim
authors.affiliation: Microbiology and Botany Department, Faculty of Science, Zagazig University, Zagazig 44519, Egypt.
authors.forename: Marwa A
authors.initials: MA
authors.lastname: Yassin
authors.affiliation: Microbiology and Botany Department, Faculty of Science, Zagazig University, Zagazig 44519, Egypt.
authors.forename: Salah E
authors.initials: SE
authors.lastname: Abdel-Ghany
authors.affiliation: Department of Nanoengineering, University of California, San Diego, UCSD, CA, USA.
authors.forename: Sadik
authors.initials: S
authors.lastname: Esener
authors.affiliation: Mid-Florida Research and Education Center, University of Florida/ Institute of Food and Agricultural Sciences, Apopka, FL, USA.
authors.forename: Gul Shad
authors.initials: GS
authors.lastname: Ali
mesh_terms: D001232:Aspergillus fumigatus / Q000201:enzymology*; D003542:Cystathionine gamma-Lyase / Q000737:chemistry* / Q000378:metabolism*; D004795:Enzyme Stability; D005656:Fungal Proteins / Q000737:chemistry* / Q000378:metabolism*; D006801:Humans; D008958:Models, Molecular; D056004:Molecular Dynamics Simulation; D011487:Protein Conformation; D020836:Protein Structure, Quaternary; D017434:Protein Structure, Tertiary; D012997:Solvents; D055672:Static Electricity; D013379:Substrate Specificity; D014364:Tryptophan / Q000737:chemistry
chemical_list: D005656:Fungal Proteins; D012997:Solvents; D014364:Tryptophan; D003542:Cystathionine gamma-Lyase
keywords: Conformational structure; Cystathionine γ-lyase; Electrostatics; MMPBSA; Molecular dynamics; Thermal shift assay