The role of the GGGX motif in determining the activity and enantioselectivity of pig liver esterase towards tertiary alcohols

Abstract

The GGG(A)X motif has been reported to determine the activity of esterases or lipases towards tertiary alcohols. The γ -isoenzyme of pig liver esterase ( γ -PLE, PLE 1) contains a GGGX motif; this motif was systematically mutated to the corresponding AGGX, GAGX, GGAX, AAAX, AAGX, AGAX and GAAX motifs to investigate its infl uence on the activity and especially enantioselectivity in the kinetic resolution of three acetates of tertiary alcohols. None of the mutants showed higher enantioselectivity than the starting enzyme ( E ϭ 17), and mutation of the central glycine to alanine completely destroyed activity and selectivity. Hence, only subtle changes of this crucial motif lead to substantial changes in esterase properties. These fi ndings were analyzed and interpreted by computer modeling.

Use of ‘small but smart’ libraries to enhance the enantioselectivity of an esterase from Bacillus stearothermophilus towards tetrahydrofuran‐3‐yl acetate

“…Three of the selected positions are close to the catalytic triad (Ser94, His223, and Asp193), and, according to previous work , are capable of affecting the activity and selectivity of the enzyme. The substrate enantiomers also interact with amino acids at positions 23–27, but these amino acids are on a sharp turn, which forms the oxyanion hole, and are responsible for stabilization of the tetrahedral intermediate . More specifically, the carboxylic oxygen of the substrate forms two hydrogen bonds with the backbone nitrogen atoms of Phe25 and Leu95 (Fig.…”

Role of amino acid residues involved in the active cavity of proline iminopeptidase in catalytic activity

“…Bulgaricus, in which mutation of the corresponding sites by chemical mutagenesis resulted in loss of catalytic activity [26]. In the other members from the α/β fold hydrolase family, such as esterase and acetylcholinesterase, mutation of the corresponding two Gly residues also had dead effect on the activity [27,28]. Furthermore, mutation of Gly42 and Cys45 also yield a profound negative influence on catalytic activity.…”

Kinetic resolution of esters from secondary and tertiary benzylic propargylic alcohols by an improved esterase-variant from Bacillus sp. BP-7

“…Esterases containing the more common GX motif in the oxyanion hole pocket do not show activity towards esters of sterically demanding tertiary alcohols because they bear too small active sites [4]. On the contrary, the configuration of the oxyanion hole in GGGX hydrolases differs considerably, thus allowing the accommodation of more bulky alcohols [23]. A schematic view of both conformations developed from a computer model was designed by Henke et al [24].…”

“…Aiming to understand the molecular mechanism of enantiorecognition of esters from tertiary alcohols by mutant EstBP7-AGA, as well as to explore further substrate diversity, we also investigated the enantioselectivity of our promising esterase variant towards a wide range of esters from chiral benzylic propargylic alcohols 1-12 (Scheme 1). Importantly, the scope of this analysis was restricted by the stability of the starting substrates, since esters of tertiary benzylic alcohols containing electron-rich substituents in the aromatic ring are prone to competing hydrolysis under the experimental conditions due to the stability of the corresponding carbocations [23]. The introduction of the strong electron withdrawing trifluoromethyl group, CF 3 , minimized this shortcoming in acetate esters 5-9, whereas related methyl derivatives 10-12 and esters 1-4 from secondary alcohols proved to be stable enough to be analyzed safely.…”