title: Stability mechanisms of a thermophilic laccase probed by molecular dynamics.
abstract: Laccases are highly stable, industrially important enzymes capable of oxidizing a large range of substrates. Causes for their stability are, as for other proteins, poorly understood. In this work, multiple-seed molecular dynamics (MD) was applied to a Trametes versicolor laccase in response to variable ionic strengths, temperatures, and glycosylation status. Near-physiological conditions provided excellent agreement with the crystal structure (average RMSD ∼0.92 Å) and residual agreement with experimental B-factors. The persistence of backbone hydrogen bonds was identified as a key descriptor of structural response to environment, whereas solvent-accessibility, radius of gyration, and fluctuations were only locally relevant. Backbone hydrogen bonds decreased systematically with temperature in all simulations (∼9 per 50 K), probing structural changes associated with enthalpy-entropy compensation. Approaching T opt (∼350 K) from 300 K, this change correlated with a beginning "unzipping" of critical β-sheets. 0 M ionic strength triggered partial denucleation of the C-terminal (known experimentally to be sensitive) at 400 K, suggesting a general salt stabilization effect. In contrast, F(-) (but not Cl(-)) specifically impaired secondary structure by formation of strong hydrogen bonds with backbone NH, providing a mechanism for experimentally observed small anion destabilization, potentially remedied by site-directed mutagenesis at critical intrusion sites. N-glycosylation was found to support structural integrity by increasing persistent backbone hydrogen bonds by ∼4 across simulations, mainly via prevention of F(-) intrusion. Hydrogen-bond loss in distinct loop regions and ends of critical β-sheets suggest potential strategies for laboratory optimization of these industrially important enzymes.
authors: Department of Chemistry, Technical University of Denmark, Kongens Lyngby, Denmark.
authors: Niels J
authors: NJ
authors: Christensen
authors: Kasper P
authors: KP
authors: Kepp
mesh_terms: D018360:Crystallography, X-Ray; D004795:Enzyme Stability; D005656:Fungal Proteins / Q000737:chemistry* / Q000235:genetics; D006031:Glycosylation; D006358:Hot Temperature; D006860:Hydrogen Bonding; D042845:Laccase / Q000737:chemistry* / Q000235:genetics; D056004:Molecular Dynamics Simulation; D016297:Mutagenesis, Site-Directed; D009994:Osmolar Concentration; D017433:Protein Structure, Secondary; D055672:Static Electricity; D013816:Thermodynamics; D055454:Trametes / Q000737:chemistry* / Q000201:enzymology
chemical_list: D005656:Fungal Proteins; D042845:Laccase
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