Molecular and Spectroscopic Characterization of Aspergillus flavipes and Pseudomonas putida L-Methionine γ-Lyase in
Ashraf S A El-Sayed1,2, Laura E Ruff3, Salah E Abdel Ghany4
1Microbiology Department, Faculty of Science, Zagazig University, Zagazig, 44519, Egypt. ashrafzu@ufl.edu.
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.
Insights
Aspergillus flavipes L-methionine γ-lyase (AfMGL) shows comparable stability and functional properties to Pseudomonas putida L-methionine γ-lyase (PpMGL), suggesting AfMGL as a promising alternative anticancer agent with improved biochemical characteristics.
Area of Science:
- Biochemistry
- Enzymology
- Structural Biology
Background:
- Pseudomonas putida L-methionine γ-lyase (PpMGL) is an effective anticancer agent, but faces challenges with antigenicity and stability.
- Aspergillus flavipes L-methionine γ-lyase (AfMGL) presents potentially superior biochemical properties compared to PpMGL.
Purpose of the Study:
- To conduct a comparative assessment of the functional properties of AfMGL and PpMGL.
- To evaluate the stability of the internal aldimine linkage, pyridoxal 5'-phosphate (PLP) tautomerism, and structural stability in response to physicochemical factors.
Main Methods:
- Comparative analysis of enzyme stability using hydroxylamine and human serum albumin.
- Assessment of enzyme structural stability and PLP tautomerism under varying pH conditions.
- Fluorescence spectroscopy to analyze intrinsic tryptophan and Schiff base.
- Differential Scanning Fluorimetry (DSF) to determine melting temperatures (Tm) in the presence of stabilizers/destabilizers.
Main Results:
- Both AfMGL and PpMGL exhibit similar stability of their internal Schiff base linkage.
- Acidic pH leads to significant cleavage of the Schiff base and enzyme unfolding, while neutral-alkaline pHs are less disruptive.
- Identical fluorescence emission spectra were observed for both enzymes at specific wavelengths.
- The melting temperatures (Tm) of AfMGL and PpMGL are high (73.1°C and 74.4°C, respectively) and slightly increased by trehalose and EDTA, but decreased by guanidine HCl and urea.
- Active sites and PLP-binding domains are conserved between AfMGL and PpMGL.
Conclusions:
- AfMGL demonstrates comparable biochemical and functional properties to PpMGL, including similar stability and conserved active site structures.
- AfMGL represents a viable alternative to PpMGL for potential clinical applications, offering improved biochemical characteristics.
- Understanding the stability and PLP tautomerism under various conditions is crucial for optimizing the use of these enzymes.
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