Determinants and Prediction of Esterase Substrate Promiscuity Patterns

Abstract

Esterases receive special attention because of their wide distribution in biological systems and environments and their importance for physiology and chemical synthesis. The prediction of esterases' substrate promiscuity level from sequence data and the molecular reasons why certain such enzymes are more promiscuous than others remain to be elucidated. This limits the surveillance of the sequence space for esterases potentially leading to new versatile biocatalysts and new insights into their role in cellular function. Here, we performed an extensive analysis of the substrate spectra of 145 phylogenetically and environmentally diverse microbial esterases, when tested with 96 diverse esters. We determined the primary factors shaping their substrate range by analyzing substrate range patterns in combination with structural analysis and protein-ligand simulations. We found a structural parameter that helps rank (classify) the promiscuity level of esterases from sequence data at 94% accuracy. This parameter, the active site effective volume, exemplifies the topology of the catalytic environment by measuring the active site cavity volume corrected by the relative solvent accessible surface area (SASA) of the catalytic triad. Sequences encoding esterases with active site effective volumes (cavity volume/SASA) above a threshold show greater substrate spectra, which can be further extended in combination with phylogenetic data. This measure provides also a valuable tool for interrogating substrates capable of being converted. This measure, found to be transferred to phosphatases of the haloalkanoic acid dehalogenase superfamily and possibly other enzymatic systems, represents a powerful tool for low-cost bioprospecting for esterases with broad substrate ranges, in large scale sequence data sets.

Relationships between Substrate Promiscuity and Chiral Selectivity of Esterases from Phylogenetically and Environmentally Diverse Microorganisms

“…They included esters with variation in size of acyl and alcohol groups and with growing residues (aromatic, aliphatic, branched, and unbranched), halogenated esters, sugar esters, lactones, an alkyl di-ester, and 20 chiral esters (including (R) and (S) enantiomers of menthyl acetate, N-benzyl-proline ethyl ester, methyl mandelate, ethyl 4-chloro-3-hydroxybutyrate, methyl 3-hydroxybutyrate, methyl 3-hydroxyvalerate, neomenthyl acetate, methyl and ethyl lactate, and pantolactone). By meaning of the partitioning coefficient (log p value), which reflects electronic and steric effects and hydrophobic and hydrophilic characteristics, the 96 esters do show a broad chemical and structural variability [5]. This chemical variability also characterized the chiral esters tested (Figure 1).…”

“…Here, we investigate the relationships between the level of substrate promiscuity and chiral selectivity of a large set of 145 phylogenetically and environmentally diverse microbial esterases, whose specific activity against 96 distinct esters that included 20 chiral esters have been recently reported [5]. We provide unambiguous experimental evidence suggesting a negative association between substrate specificity and chiral selectivity in native esterases.…”

“…We have recently described an extensive analysis of the substrate spectra of 145 phylogenetically and environmentally diverse microbial esterases [5]. Experimental data on substrate conversion (i.e., units g −1 or U·g −1 ) followed for 24 h, at pH 8.0 and 30 • C was reported for 96 distinct esters.…”

“…Nature is a rich reservoir from where enzymes can be isolated [3,4], because they are continuously evolving as a consequence of natural selection. Promiscuous enzymes are effective for converting multiple substrates, thus, they are industrially relevant [4][5][6]. Enzymes need to also be robust and, preferably, chiral selective to reduce raw material costs in the synthesis of pure chiral compounds [1,2,4,7].…”

“…The activity of these esterases relies mainly on a catalytic triad usually formed by Ser, Asp/Glu, and His [8]. This enzyme class was selected for a number of reasons: it is widely distributed in the environment, it has important physiological functions, it includes hydrolases that are among the most important industrial biocatalysts, and extensive biochemical knowledge has been accumulated [4,5,7].…”