Shift in Bacillus sp. JR3 esterase LipJ activity profile after addition of essential residues from family I.5 thermophilic lipases

Abstract

We recently described LipJ, a mesophilic esterase clustering in a specific clade of bacterial lipase family I.5, bearing the same structure but several distinct sequence motifs. Two mutagenesis approaches were used here to modify LipJ by addition of common features of family 1.5 lipases, searching for changes on the activity profile. Iterative saturation mutagenesis of a residue possibly involved in Zn2+ coordination produced a LipJ variant –H110N– displaying a remarkable shift in substrate specificity, possibly boosted by changes in number and distance of relevant polar interactions for Zn2+ coordination. Also, variants E27 A, G136 A and M139Q were obtained by site directed mutagenesis as single, double or triple mutants, showing a general shift in specificity towards higher temperature and longer chain-length substrates, all of them displaying higher activity than LipJ on C7 substrates. The results obtained suggest that the N-terminal region of LipJ may indeed contribute to substrate accommodation, whereas mutations at the pentapeptide displayed maximum activity at 50 °C, with the triple mutant showing activity even at 80 °C, greatly enhanced by Mn2+. 3D modelling and molecular docking analysis of selected mutants provided clues for a better accommodation of medium chain-length substrates, and for a modified Zn2+ environment, in agreement with the substrate and temperature profile shifts observed.