The Role of Arg157Ser in Improving the Compactness and Stability of ARM Lipase

Abstract

Consensus approach is an efficient strategy to identify hot residue important for compactness and stability of protein. Structure of ARM lipase was modeled to explore the possible effect of critical point mutation towards structure and function. The significant difference of amino acid at position 157 between ARM lipase (Arg157) and other thermostable lipases (Ser157) was targeted as a critical residue. Using YASARA software, Arg157 was substituted to Ser and subsequently the energy minimized. Both ARM and R157S lipases were analyzed by MD simulations at different temperatures (50ºC,60ºC, and 70ºC). MD simulation result showed that R157S lipase had lower value of RMSD, RMSF, solvent accessible surface area (SASA) and radius of gyration than native ARM lipase. It indicated that R157S lipase had higher compactness in the structure leading to enhanced stability. To validate the computational data, the substitution of Arg157 to Ser has been conducted using site-directed mutagenesis experimentally. The catalytic efficiency (k cat /K M ) of R157S lipase was refold better than ARM lipase of 70°C. Circular dichorism study revealed that R157S lipase had increased thermostability with higher T m value (71.6°C) than its wildtype (63.9°C) indicating a better compactness as revealed by spectrofluorocence study. Thus the rational design of substituting Arg157 with Ser improved the protein folding of mutant lipase as shown in MD simulations and subsequently increased the catalytic effectiveness and thermodynamic stability. during evolution. The substitution of critical residue was analyzed using in silico to avoid unnecessary conformational distortion in engineered lipase and validate with kinetic study and thermodynamic study. Methods Preparation of 3D structures of ARM and R157S lipasesThe 3D structure of ARM lipase was predicted by comparative modeling. The first step in comparative modeling is the assignment of the unknown ARM lipase structure to a protein family with thousands of sequences already stored in protein databases (PDB). The structure of ARM lipase was modeled using available software packages such as HOMOLOGY and MODELLER, Accelrys, San Diego (http://www. accelrys.com). The unknown ARM lipase structure (target protein) was built from the known 3D structure of T1 lipase (2DSN) as a template. The final model of ARM lipase was evaluated using Ramachandran plot. Using YASARA, the three-dimensional structure of the mutant (R157S lipase) was modeled by substituting Arg157 to Ser and energy minimization was used to release bad contact. MD simulations of ARM and R157S lipasesMD simulations were performed on ARM lipase and R157S lipase structures using Amber03 force field in YASARA package. The periodic box was filled with explicit water molecules to a density of 0.98320 g/l. The whole system was neutralized by adding counter ions and all ionizable protein groups were protonated according to their tabulated pKa values at pH 7 of the medium. Water molecules were relaxed by simulated annealing procedure. Minimiza...

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The Influence of Calcium toward Order/Disorder Conformation of Repeat-in-Toxin (RTX) Structure of Family I.3 Lipase from Pseudomonas fluorescens AMS8

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