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.
Highlights
Laccases are multicopper oxidases (MCO) [1,2], found in fungi, bacteria, plants, and insects, with exquisite temperature- and pHtolerance and capable of oxidizing a wide array of organic and inorganic substrates [3,4]
This paper reports an investigation of the molecular drivers of the thermostability of the widely explored a-isoform of Trametes versicolor laccases (TvL) [47], denoted here TvLa, using a multiple-seed approach designed to reduce systematic molecular dynamics (MD) errors and monitor structural response under conditions resembling more an experimental assay optimization, notably T, ionic strength (IS), and glycosylation status, instead of the usual fixed conditions of standard MD simulations
We identify several determinants of thermostability in the protein, including i) a small anion (F2) destabilization effect; ii) a glycosylation effect on secondary-structure persistence, and iii) a quantification and residual localization of secondary-structure interactions sacrificed as a result of the entropy-enthalpy compensation at high T, with ca. nine persistent backbonebackbone hydrogen bonds lost per 50-K increase
Summary
Laccases are multicopper oxidases (MCO) [1,2], found in fungi, bacteria, plants, and insects, with exquisite temperature- and pHtolerance and capable of oxidizing a wide array of organic and inorganic substrates [3,4]. Due to their clean reactions having only water as byproduct, they are very attractive as industrial biocatalysts [5,6]. They have attracted considerable interest due to their special structure-function correlations, notably the tuning of the high redox potentials and the oxygen activation mechanism that reduces dioxygen to produce water [1,7]. One electron is sequentially abstracted from each of four substrate molecules at the T1 site and transported to the T2 and T3 sites where dioxygen is reduced to two water molecules [1,7]
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