Biochemical Stability and Molecular Dynamic Characterization of Aspergillus fumigatus Cystathionine γ-Lyase in Response to Various Reaction Effectors

Handle URI:
http://hdl.handle.net/10754/567038
Title:
Biochemical Stability and Molecular Dynamic Characterization of Aspergillus fumigatus Cystathionine γ-Lyase in Response to Various Reaction Effectors
Authors:
El-Sayed, Ashraf S.A.; Abdel-Azeim, Safwat ( 0000-0001-8611-1251 ) ; Ibrahim, Hend M.; Yassin, Marwa A.; Abdel-Ghany, Salah E.; Esener, Sadik; Ali, Gul Shad
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.
KAUST Department:
KAUST Catalysis Center (KCC)
Citation:
Biochemical Stability and Molecular Dynamic Characterization of Aspergillus fumigatus Cystathionine γ-Lyase in Response to Various Reaction Effectors 2015 Enzyme and Microbial Technology
Publisher:
Elsevier BV
Journal:
Enzyme and Microbial Technology
Issue Date:
11-Aug-2015
DOI:
10.1016/j.enzmictec.2015.08.004
Type:
Article
ISSN:
01410229
Additional Links:
http://linkinghub.elsevier.com/retrieve/pii/S0141022915300399
Appears in Collections:
Articles; KAUST Catalysis Center (KCC)

Full metadata record

DC FieldValue Language
dc.contributor.authorEl-Sayed, Ashraf S.A.en
dc.contributor.authorAbdel-Azeim, Safwaten
dc.contributor.authorIbrahim, Hend M.en
dc.contributor.authorYassin, Marwa A.en
dc.contributor.authorAbdel-Ghany, Salah E.en
dc.contributor.authorEsener, Sadiken
dc.contributor.authorAli, Gul Shaden
dc.date.accessioned2015-08-16T13:09:09Zen
dc.date.available2015-08-16T13:09:09Zen
dc.date.issued2015-08-11en
dc.identifier.citationBiochemical Stability and Molecular Dynamic Characterization of Aspergillus fumigatus Cystathionine γ-Lyase in Response to Various Reaction Effectors 2015 Enzyme and Microbial Technologyen
dc.identifier.issn01410229en
dc.identifier.doi10.1016/j.enzmictec.2015.08.004en
dc.identifier.urihttp://hdl.handle.net/10754/567038en
dc.description.abstractCystathionine 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.en
dc.language.isoenen
dc.publisherElsevier BVen
dc.relation.urlhttp://linkinghub.elsevier.com/retrieve/pii/S0141022915300399en
dc.rightsNOTICE: this is the author’s version of a work that was accepted for publication in Enzyme and Microbial Technology. Changes resulting from the publishing process, such as peer review, editing, corrections, structural formatting, and other quality control mechanisms may not be reflected in this document. Changes may have been made to this work since it was submitted for publication. A definitive version was subsequently published in Enzyme and Microbial Technology, 11 August 2015. DOI: 10.1016/j.enzmictec.2015.08.004en
dc.subjectAspergillus fumigatusen
dc.subjectCystathionine γ-lyaseen
dc.subjectConformational structureen
dc.subjectElectrostaticsen
dc.subjectMolecular dynamicsen
dc.subjectMMPBSAen
dc.subjectThermal shift assayen
dc.titleBiochemical Stability and Molecular Dynamic Characterization of Aspergillus fumigatus Cystathionine γ-Lyase in Response to Various Reaction Effectorsen
dc.typeArticleen
dc.contributor.departmentKAUST Catalysis Center (KCC)en
dc.identifier.journalEnzyme and Microbial Technologyen
dc.eprint.versionPost-printen
dc.contributor.institutionMicrobiologyand Botany Department, Faculty of Science, Zagazig University, Zagazig, 44519, Egypten
dc.contributor.institutionMid-Florida Research and Education Center, University of Florida/ Institute of Food and Agricultural Sciences, Apopka, FL, USAen
dc.contributor.institutionDepartment of Medical Biochemistry and Molecular Biology, Faculty of Medicine, Zagazig University, Zagazig, 44519, Egypten
dc.contributor.institutionDepartment of Nanoengineering, University of California, San Diego, UCSD, CA, USAen
dc.contributor.affiliationKing Abdullah University of Science and Technology (KAUST)en
kaust.authorAbdel-Azeim, Safwaten
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