Esterases receive special attention because 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 ranking (classifying) 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 datasets.
The resolution of methyl (+/-)-3-hydroxypentanoate catalysed by Candida antarctica lipase B has been performed by using ammonia and benzyl amine as nucleophiles. In all cases, the lipase reacts faster with the R enantiomer of the ester, but when benzyl amine is used, the enantiomeric ratio is approximately three times as high as that measured for ammonia. The analysis of the molecular dynamics simulations carried out over the corresponding deacylation transition state analogues indicated specular binding modes between enantiomers that vary greatly upon the nucleophile used. For the case of ammonia, an intramolecular hydrogen bond between the beta-hydroxyl group and the protons of the nucleophile is established. However, the presence of the substituent in benzyl amine disrupts this interaction. Instead, the acyl chain binds to a more restrictive area of the protein where the higher number of contacts established with the side chains of Thr40, Gln157 and Ile189 have been identified as the reason for the higher enantioselectivity observed in the aminolysis reaction.
