ABSTRACT
Metagenomics offers the possibility to screen for versatile biocatalysts. In this study, the microbial community of the Sorghum bicolor rhizosphere was spiked with technical cashew nut shell liquid, and after incubation, the eDNA was extracted and subsequently used to build a metagenomic library. We report the biochemical features and crystal structure of a novel esterase from the family IV, EH0, retrieved from an uncultured sphingomonad after a functional screen in tributyrin agar plates. EH0 (Topt, 50 °C; Tm, 55.7 °C; pHopt, 9.5) was stable in the presence of 10-20% v/v organic solvents and exhibited hydrolytic activity against p-nitrophenyl esters from acetate to palmitate, preferably butyrate (496 U mg−1), and a large battery of 69 structurally different esters (up to 30.2 U mg−1), including bis(2-hydroxyethyl)-terephthalate (0.16 ± 0.06 U mg−1). This broad substrate specificity contrasts with the fact that EH0 showed a long and narrow catalytic tunnel, whose access appears to be hindered by a thigth folding of its cap domain. We propose that this cap domain is a highly flexible structure whose opening is mediated by unique structural elements, one of which is the presence of two contiguous proline residues likely acting as possible hinges, that altogether allow for the entrance of the substrates. Therefore, this work provides a new role for the cap domain, which until now was thought to be immobile elements that contain hydrophobic patches involved in substrate pre-recognition and in turn substrate specificity within family IV esterases.
IMPORTANCE A better understanding of structure–function relationships of enzymes allows revealing key structural motifs or elements. Here, we studied the structural basis of the substrate promiscuity of EH0, a family IV esterase, isolated from a sample of the Sorghum bicolor rhizosphere microbiome exposed to technical cashew nut shell liquid. The analysis of EH0 revealed the potential of the sorghum rhizosphere microbiome as a source of enzymes with interesting properties, such as pH and solvent tolerance and remarkably broad substrate promiscuity. Its structure resembled those of homologous proteins from mesophilic Parvibaculum and Erythrobacter spp. and hyperthermophilic Pyrobaculum and Sulfolobus spp. and had a very narrow, single-entry access tunnel to the active site, access which is controlled by a capping domain that includes a number of not conserved proline residues. These structural markers, distinct from those of other substrate promiscuous esterases, can help tuning substrate profiles beyond tunnel and active site engineering.
Competing Interest Statement
The authors have declared no competing interest.
Footnotes
This manuscript is an updated version of the previous one, which contains the following additional works and clarified the following main points: a) We performed additional solvent-activity tests to ensure that the concentrations used for determining the specific activity did not introduce any bias. b) We have better clarified the methods for activity determination. c) We have introduced changes along the manuscript to clarify the significance of the structural findings. d) We have compared the substrate specificity of EH0 with that of other family IV members tested with the same set of ester substrates and discussed the results from a structural point of view. e) We have now provided additional information about the reaction kinetics, raw data and calculations that denote high robustness, sensitivity, precision and reproducibility on the determinations.
