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.

Optimization of Laccase Fermentation and Evaluation of Kinetic and Thermodynamic Parameters of a Partially Purified Laccase Produced byDaedalea flavida

“…At an optimal pH, maximum stability of the laccase could be contributed by multiple factors, that is, backbone hydrogen bonds, optimal salt-bridge structures, and polysaccharide residues. [53] However, it is difficult to explain their individual contributions when the pH deviates from optimal pH. Above 30 C, the optimal pH shifts to 5.7 where DG Ã is maximum.…”

Investigating the Role of Conformational Effects on Laccase Stability and Hyperactivation under Stress Conditions

“…Particular attention was paid to the simulation of the copper ions, as they are involved in the ETP, and distortions of ETP have been postulated to be involved in the inactivation and biological turnover of the enzyme . In previous MD simulations the ion coordination had been treated by defining fix bonds for constraining their relative positions; this makes MD simulations of limited use for describing phenomena affecting the ETP. Therefore, the coppers ions were modeled by defining each coordination sphere as pair of interactions (for details see the Experimental Section).…”

DESIGN OF <i>CANDIDA ANTARCTICA</i> LIPASE B THERMOSTABILITY IMPROVEMENT BY INTRODUCING EXTRA DISULFIDE BOND INTO THE ENZYME

“…As we can see, molecular dynamic simulation is still the most effective way to model the structure and reactivity of an enzyme (Gabel et al, 2009;Lousa et al, 2012;Christensen and Kepp, 2013). In this respect, we have utilized this method since 2011 (Tambunan et al, 2011a;2011b;Parikesit et al, 2013).…”