Molecular mechanism of the hydration of Candida antarctica lipase B in gas phase: water adsorption isotherms and molecular dynamics simulations Ricardo J. F. Branco ‡ , Marianne Graber † , Vinciane Denis † and Jürgen Pleiss ‡* ‡ Institute of Technical Biochemistry, University of Stuttgart, Allmandring 31, 70569 Stuttgart, Germany. † Université de la Rochelle, Pôle Sciences et Technologie -Bât. Marie Curie, UMR6250 LIENSs CNRS-ULR, Avenue Michel Crépeau, 17042 La Rochelle, Cedex 01, France Dedicated to Professor Karl Hult on the occasion of his 65 th birthday * Communicating author: Prof. Dr. Jürgen Pleiss Institute of Technical Biochemistry University of Stuttgart Allmandring 31 D-70569 Stuttgart Germany E-mail: [email protected] Phone: (+49) 711-68563191 1 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 1
ABSTRACT Hydration is a major determinant of activity and selectivity of enzymes in organic solvents or in gas phase. The molecular mechanism of the hydration of Candida antarctica lipase B (CALB) and its dependence on the thermodynamic activity of water a w was studied by molecular dynamics simulations and compared to experimentally determined water sorption isotherms. Hydration occurred in two phases. At low water activity, single water molecules bound to specific water binding sites at the protein surface. As the water activity increased, water networks gradually developed. The number of protein-bound water increased linearly with a w , until at a w = 0.5 a spanning water network was formed consisting of 311 water molecules which covered the hydrophilic surface of CALB, with the exception of the hydrophobic substrate binding site. At higher water activity, the thickness of the hydration shell increased up to 10 Å close to a w = 1. Above a limit of 1600 protein-bound water molecules the hydration shell becomes unstable and the formation of pure water droplets occurs in this oversaturated simulation conditions. While the structure and the overall flexibility of CALB was independent of the hydration state, the flexibility of individual loops was sensitive to hydration: some loops such as part of the substrate binding site became more flexible, while other parts of the protein became more rigid upon hydration. However, the molecular mechanism of how flexibility is related to activity and selectivity is still elusive. Keywords : thermodynamic activity of water hydration mechanism molecular modeling deglycosylation 2 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 2
INTRODUCTION Candida antarctica lipase B (CALB) is an efficient catalyst for hydrolysis of ester substrates in water and esterification in organic solvents. It is used in many industrial applications because of its high enantioselectivity, wide range of substrates, thermal stability, and stability in organic solvents.  CALB belongs to the α/β hydrolase fold family with a conserved catalytic triad consisting of Ser, His, and Asp/Glu.
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