The mobility of the cap domain is essential for the substrate promiscuity of a family IV esterase from sorghum rhizosphere microbiome

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; Td, 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 (496.5 U mg-1), and a large battery of 69 structurally different esters (up to 30.23 U mg-1), including bis(2-hydroxyethyl)-terephthalate (0.16 ± 0.062 U mg-1). This broad substrate ambiguity contrasts with the fact that EH0 showed a long and narrow catalytic tunnel, whose access appears to be hindered by a cap domain. We demonstrated that this cap domain is a mobile element whose opening is controlled by two contiguous proline residues which, likely acting as possible hinges, allows for the entrance of the substrates. Together, 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 ambiguity within family IV esterases.