1. Metal binding to cutinase-like enzyme from Saccharomonospora viridis AHK190 and its effects on enzyme activity and stability
- Author
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Fusako Kawai, Yuki Kimura, Gert-Jan Bekker, Akane Senga, Masayuki Oda, Yoshiji Hantani, and Narutoshi Kamiya
- Subjects
Models, Molecular ,inorganic chemicals ,Cutinase ,Circular dichroism ,Stereochemistry ,Metal ions in aqueous solution ,Biochemistry ,03 medical and health sciences ,Enzyme Stability ,Magnesium ,Thermal stability ,Binding site ,Molecular Biology ,030304 developmental biology ,chemistry.chemical_classification ,Manganese ,0303 health sciences ,Binding Sites ,biology ,Chemistry ,030302 biochemistry & molecular biology ,Isothermal titration calorimetry ,General Medicine ,Enzyme assay ,Actinobacteria ,Zinc ,Enzyme ,Mutation ,biology.protein ,Thermodynamics ,Calcium ,Carboxylic Ester Hydrolases - Abstract
A cutinase from Saccharomonospora viridis AHK190, Cut190, can hydrolyze polyethylene terephthalate and has a unique feature that the activity and stability are regulated by Ca2+ binding. Our recent structural and functional analyses showed three Ca2+ binding sites and their respective roles. Here, we analysed the binding thermodynamics of Mn2+, Zn2+ and Mg2+ to Cut190 and their effects on the catalytic activity and thermal stability. The binding affinities of Mn2+ and Zn2+ were higher than that of Mg2+ and are all entropy driven with a binding stoichiometry of three, one and one for Zn2+, Mn2+ and Mg2+, respectively. The catalytic activity was measured in the presence of the respective metals, where the activity of 0.25 mM Mn2+ was comparable to that of 2.5 mM Ca2+. Our 3D Reference Interaction Site Model calculations suggested that all the ions exhibited a high occupancy rate for Site 2. Thus, Mn2+ and Mg2+ would most likely bind to Site 2 (contributes to stability) with high affinity, while to Sites 1 and 3 (contributes to activity) with low affinity. We elucidate the metal-dependent structural and functional properties of Cut190 and show the subtle balance on structure stability and flexibility is controlled by specific metal ions.
- Published
- 2019