Engineering the pH-optimum of a triglyceride lipase: from predictions based on electrostatic computations to experimental results.
The optimisation of enzymes for particular purposes or conditions remains an important target in virtually all protein engineering endeavours. Here, we present a successful strategy for altering the pH-optimum of the triglyceride lipase cutinase from Fusarium solani pisi. The computed electrostatic pH-dependent potentials in the active site environment are correlated with the experimentally observed enzymatic activities. At pH-optimum a distinct negative potential is present in all the lipases and esterases that we studied so far. This has prompted us to propose the "The Electrostatic Catapult Model" as a model for product release after cleavage of the ester bond. The origin of the negative potential is associated with the titration status of specific residues in the vicinity of the active site cleft. In the case of cutinase, the role of Glu44 was systematically investigated by mutations into Ala and Lys. Also, the neighbouring Thr45 was mutated into Proline, with the aim of shifting the spatial location of Glu44. All the charge mutants displayed altered titration behaviour of active site electrostatic potentials. Typically, the substitution of the residue Glu44 pushes the onset of the active site negative potential towards more alkaline conditions. We, therefore, predicted more alkaline pH optima, and this was indeed the experimentally observed. Finally, it was found that the pH-dependent computed Coulombic energy displayed a strong correlation with the observed melting temperatures of native cutinase.
Neves-Petersen
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MT
The Biostructure and Protein Engineering Group, Department of Life Sciences, Aalborg University, Sohngaardsholmsvej 49, DK-9000 Aalborg, Denmark.
Petersen
E I
EI
Fojan
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Noronha
M
M
Madsen
R G
RG
Petersen
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SB
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001483:Base Sequence; D002265:Carboxylic Ester Hydrolases / Q000235:genetics / Q000378:metabolism; D002942:Circular Dichroism; D003198:Computer Simulation; D004795:Enzyme Stability; D005670:Fusarium / Q000201:enzymology; D006863:Hydrogen-Ion Concentration; D008049:Lipase / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D008956:Models, Chemical; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D015202:Protein Engineering / Q000379:methods*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D055672:Static Electricity
D011994:Recombinant Proteins; D002265:Carboxylic Ester Hydrolases; C019952:cutinase; D008049:Lipase