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.
Title: Direct enzymatic acylation of cellulose pretreated in BMIMCl ionic liquid Gremos S, Zarafeta D, Kekos D, Kolisis F Ref: Bioresour Technol, 102:1378, 2011 : PubMed
Cellulose esters are an important class of functional biopolymers with great interest in the chemical industry. In this work the enzymatic acylation of Avicel cellulose with vinyl propionate, vinyl laurate and vinyl stearate, has been performed successfully in a solvent free reaction system. At first cellulose was putted into the ionic liquid BMIMCl (1-n-butyl-3-methylimidazolium chloride) in order to facilitate the unwrap of the structure of the polysaccharide molecule and make it accessible to the enzyme. Thus, after this pretreatment the enzymatic esterification reaction was performed using various hydrolases. The enzymes capable of catalyzing the acylation of cellulose were found to be the immobilized esterase from hog liver and the immobilized cutinase from Fusarium solani, while the lipases used did not show any catalytic activity. Cellulose esters of propionate, laurate and stearate were synthesized with a degree of esterification of 1.9%, 1.3% and 1.0%, respectively. It is the first successful direct enzymatic acylation of cellulose with long chain fatty acids.
Title: Non-lipolytic and lipolytic sequence-related carboxylesterases: a comparative study of the structure-function relationships of rabbit liver esterase 1 and bovine pancreatic bile-salt-activated lipase Chahinian H, Fantini J, Garmy N, Manco G, Sarda L Ref: Biochimica & Biophysica Acta, 1801:1195, 2010 : PubMed
To differentiate esterases from lipases at the structure-function level, we have compared the kinetic properties and structural features of sequence-related esterase 1 from rabbit liver (rLE) and bile-salt-activated lipase from bovine pancreas (bBAL). In contrast to rLE, bBAL hydrolyses water-insoluble medium and long chain esters as vinyl laurate, trioctanoin and olive oil. Conversely, rLE and bBAL are both active on water-soluble short chain esters as vinyl acetate, vinyl propionate, vinyl butyrate, tripropionin, tributyrin and p-nitrophenyl butyrate. However, the enzymes show distinctive kinetic behaviours. rLE displays maximal activity at low substrate concentration, below the critical micelle concentration, whereas bBAL acts preferencially on emulsified esters, at concentration exceeding the solubility limit. Comparison of the 3D structures of rLE and bBAL shows, in particular, that the peptide loop at positions 116-123 in bBAL is deleted in rLE. This peptide segment interacts with a bile salt molecule thus inducing a conformational transition which gives access to the active site. Inhibition studies and manual docking of a bulky ester molecule as vinyl laurate in the catalytic pocket of rLE and bBAL show that the inability of the esterase to hydrolyse large water-insoluble esters is not due to steric hindrance. It is hypothesized that esterases lack specific hydrophobic structures involved both in the stabilization of the lipase-lipid adsorption complex at interfaces and in the spontaneous transfer of a single substrate molecule from interface to the catalytic site.
