Dataset Information

Effects of Lid 1 Mutagenesis on Lid Displacement, Catalytic Performances and Thermostability of Cold-active Pseudomonas AMS8 Lipase in Toluene.

ABSTRACT: Pseudomonas fluorescens AMS8 lipase lid 1 structure is rigid and holds unclear roles due to the absence of solvent-interactions. Lid 1 region was stabilized by 17 hydrogen bond linkages and displayed lower mean hydrophobicity (0.596) compared to MIS38 lipase. Mutating lid 1 residues, Thr-52 and Gly-55 to aromatic hydrophobic-polar tyrosine would churned more side-chain interactions between lid 1 and water or toluene. This study revealed that T52Y leads G55Y and its recombinant towards achieving higher solvent-accessible surface area and longer half-life at 25 to 37?°C in 0.5% (v/v) toluene. T52Y also exhibited better substrate affinity with long-chain carbon substrate in aqueous media. The affinity for pNP palmitate, laurate and caprylate increased in 0.5% (v/v) toluene in recombinant AMS8, but the affinity in similar substrates was substantially declined in lid 1 mutated lipases. Regarding enzyme efficiency, the recombinant AMS8 lipase displayed highest value of kcat/Km in 0.5% (v/v) toluene, mainly with pNPC. In both hydrolysis reactions with 0% and 0.5% (v/v) toluene, the enzyme efficiency of G55Y was found higher than T52Y for pNPL and pNPP. At 0.5% (v/v) toluene, both mutants showed reductions in activation energy and enthalpy values as temperature increased from 25 to 35?°C, displaying better catalytic functions. Only T52Y exhibited increase in entropy values at 0.5% (v/v) toluene indicating structure stability. As a conclusion, Thr-52 and Gly-55 are important residues for lid 1 stability as their existence helps to retain the geometrical structure of alpha-helix and connecting hinge.

SUBMITTER: Yaacob N

PROVIDER: S-EPMC6383135 | biostudies-literature | 2019

REPOSITORIES: biostudies-literature

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Project description:Cavities are created by hydrophobic interactions between residue side chain atoms during the folding of enzymes. Redesigning cavities can improve the thermostability and catalytic activity of the enzyme; however, the synergistic effect of cavities remains unclear. In this study, Rhizomucor miehei lipase (RML) was used as a model to explore volume fluctuation and spatial distribution changes of the internal cavities, which could reveal the roles of internal cavities in the thermostability and catalytic activity. We present an inside out cavity engineering (CE) strategy based on computational techniques to explore how changes in the volumes and spatial distribution of cavities affect the thermostability and catalytic activity of the enzyme. We obtained 12 single-point mutants, among which the melting temperatures (Tm) of 8 mutants showed an increase of more than 2°C. Sixteen multipoint mutations were further designed by spatial distribution rearrangement of internal cavities. The Tm of the most stable triple variant, with mutations including T21V (a change of T to V at position 21), S27A, and T198L (T21V/S27A/T198L), was elevated by 11.0°C, together with a 28.7-fold increase in the half-life at 65°C and a specific activity increase of 9.9-fold (up to 5,828 U mg-1), one of the highest lipase activities reported. The possible mechanism of decreased volumes and spatial rearrangement of the internal cavities improved the stability of the enzyme, optimizing the outer substrate tunnel to improve the catalytic efficiency. Overall, the inside out computational redesign of cavities method could help to deeply understand the effect of cavities on enzymatic stability and activity, which would be beneficial for protein engineering efforts to optimize natural enzymes. IMPORTANCE In the present study, R. miehei lipase, which is widely used in various industries, provides an opportunity to explore the effects of internal cavities on the thermostability and catalytic activity of enzymes. Here, we execute high hydrostatic pressure molecular dynamics (HP-MD) simulations to screen the critical internal cavity and reshape the internal cavities through site-directed mutation. We show that as the global internal cavity volume decreases, cavity rearrangement can improve the stability of the protein while optimizing the substrate channel to improve the catalytic efficiency. Our results provide significant insights into understanding the mechanism of action of the internal cavity. Our strategy is expected to be applied to other enzymes to promote increases in thermostability and catalytic activity.

Dataset Information

Effects of Lid 1 Mutagenesis on Lid Displacement, Catalytic Performances and Thermostability of Cold-active Pseudomonas AMS8 Lipase in Toluene.

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