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Molecular and Spectroscopic Characterization of Aspergillus flavipes and Pseudomonas putida L-Methionine γ-Lyase in Vitro

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Abstract

Pseudomonas putida L-methionine γ-lyase (PpMGL) has been recognized as an efficient anticancer agent, however, its antigenicity and stability remain as critical challenges for its clinical use. From our studies, Aspergillus flavipes L-methionine γ-lyase (AfMGL) displayed more affordable biochemical properties than PpMGL. Thus, the objective of this work was to comparatively assess the functional properties of AfMGL and PpMGL via stability of their internal aldimine linkage, tautomerism of pyridoxal 5′-phosphate (PLP) and structural stability responsive to physicochemical factors. The internal Schiff base of AfMGL and PpMGL have the same stability to hydroxylamine and human serum albumin. Acidic pHs resulted in strong cleavage of the internal Schiff base, inducing the unfolding of MGLs, compared to neutral-alkaline pHs. At λ 280 nm excitation, both AfMGL and PpMGL have identical fluorescence emission spectra at λ 335 nm for the intrinsic tryptophan and λ 560 nm for the internal Schiff base. The maximum PLP tautomeric shift of ketoenamine to enolimine was detected at acidic pH causing complete enzyme unfolding, subunits dissociation and tautomeric shift of intrinsic PLP, rather than neutral-alkaline ones. The T m of AfMGL and PpMGL in presence of thermal stabilizer/ destabilizer was assayed by DSF. The T m of AfMGL and PpMGL was 73.1 °C and 74.4 °C, respectively, suggesting the higher proximity to the tertiary structure of both enzymes. The T m of AfMGL and PpMGL was slightly increased by trehalose and EDTA in contrast to guanidine HCl and urea. The active site and PLP-binding domains are identically conserved in both AfMGL and PpMGL.

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Authors and Affiliations

  1. Microbiology Department, Faculty of Science, Zagazig University, Zagazig, 44519, Egypt

    Ashraf S. A. El-Sayed & Salah E. Abdel Ghany

  2. MREC, Department of Plant Pathology, University of Florida, Apopka, FL, USA

    Ashraf S. A. El-Sayed & Gul Shad Ali

  3. Department of Nanoengineering, University of California, San Diego, UCSD, San Diego, CA, USA

    Laura E. Ruff & Sadik Esener

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  1. Ashraf S. A. El-Sayed
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Correspondence to Ashraf S. A. El-Sayed.

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El-Sayed, A.S.A., Ruff, L.E., Ghany, S.E.A. et al. Molecular and Spectroscopic Characterization of Aspergillus flavipes and Pseudomonas putida L-Methionine γ-Lyase in Vitro. Appl Biochem Biotechnol 181, 1513–1532 (2017). https://doi.org/10.1007/s12010-016-2299-x

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