TY - JOUR

T1 - The Mobility of the Cap Domain Is Essential for the Substrate Promiscuity of a Family IV Esterase from Sorghum Rhizosphere Microbiome

AU - Distaso, Marco

AU - Cea-Rama, Isabel

AU - Coscolín, Cristina

AU - Chernikova, Tatyana

AU - Tran, Hai

AU - Ferrer, Manuel

AU - Sanz-Aparico, Julia

AU - Golyshin, Peter

PY - 2023/1/31

Y1 - 2023/1/31

N2 - 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 environmental DNA (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, EH 0, retrieved from an uncultured sphingomonad after a functional screen in tributyrin agar plates. EH 0 (optimum temperature [ T opt], 50°C; melting temperature [ T m ], 55.7°C; optimum pH [pH opt], 9.5) was stable in the presence of 10 to 20% (vol/vol) 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 EH 0 showed a long and narrow catalytic tunnel, whose access appears to be hindered by a tight 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, which together 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 an immobile element that contained hydrophobic patches involved in substrate prerecognition and in turn substrate specificity within family IV esterases. IMPORTANCE A better understanding of structure-function relationships of enzymes allows revelation of key structural motifs or elements. Here, we studied the structural basis of the substrate promiscuity of EH 0, a family IV esterase, isolated from a sample of the Sorghum bicolor rhizosphere microbiome exposed to technical cashew nut shell liquid. The analysis of EH 0 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, with access controlled by a capping domain that includes a number of nonconserved proline residues. These structural markers, distinct from those of other substrate-promiscuous esterases, can help in tuning substrate profiles beyond tunnel and active site engineering.

AB - 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 environmental DNA (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, EH 0, retrieved from an uncultured sphingomonad after a functional screen in tributyrin agar plates. EH 0 (optimum temperature [ T opt], 50°C; melting temperature [ T m ], 55.7°C; optimum pH [pH opt], 9.5) was stable in the presence of 10 to 20% (vol/vol) 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 EH 0 showed a long and narrow catalytic tunnel, whose access appears to be hindered by a tight 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, which together 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 an immobile element that contained hydrophobic patches involved in substrate prerecognition and in turn substrate specificity within family IV esterases. IMPORTANCE A better understanding of structure-function relationships of enzymes allows revelation of key structural motifs or elements. Here, we studied the structural basis of the substrate promiscuity of EH 0, a family IV esterase, isolated from a sample of the Sorghum bicolor rhizosphere microbiome exposed to technical cashew nut shell liquid. The analysis of EH 0 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, with access controlled by a capping domain that includes a number of nonconserved proline residues. These structural markers, distinct from those of other substrate-promiscuous esterases, can help in tuning substrate profiles beyond tunnel and active site engineering.

KW - crystal structure

KW - esterase

KW - metagenome

KW - promiscuity

KW - rhizosphere

KW - rhizosphere-inhabiting microbes

KW - α/β-fold hydrolase

U2 - 10.1128/aem.01807-22

DO - 10.1128/aem.01807-22

M3 - Article

C2 - 36602332

VL - 89

SP - e0180722

JO - Applied and Environmental Microbiology

JF - Applied and Environmental Microbiology

SN - 0099-2240

IS - 1

M1 - e0180722

ER -