bioRxiv Science⌕ Search
Home/Archive/bioRxiv

bioRxiv · 10.1101/2022.09.10.507389

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

Distaso, M.·Cea-Rama, I.·Coscolin, C.·Chernikova, T. N.·Tran, H.·Ferrer, M.·Sanz-Aparicio, J.·Golyshin, P. N.
View original source ↗Export metadata ↓

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 {degrees}C; Tm, 55.7 {degrees}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. IMPORTANCEA 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.

Source connections

Explore related subjects

biochemistryPreprint

Keep this discovery

Explore connections, maps & timelines

BibTeXRIS

Distaso, M., Cea-Rama, I., Coscolin, C., Chernikova, T. N., Tran, H., Ferrer, M., Sanz-Aparicio, J., Golyshin, P. N.. 2022-09-10. The mobility of the cap domain is essential for the substrate promiscuity of a family IV esterase from sorghum rhizosphere microbiome. https://doi.org/10.1101/2022.09.10.507389

Cite the original work for its findings. Save a collection to share your selection of sources.

KEEP EXPLORING

Related preprints

bioRxiv2026

Modular assembly of mitochondrial complex III from a dynamic matrix domain pool

Mitochondrial complex III is a central component of the cellular energy metabolism. It is an obligate dimer of catalytic and accessory subunits that are encoded by two genomes. How its large matrix domain, formed by the core proteins Qcr1 and Qcr2, is built and joined to the membrane part and during assembly is not known. Here we show that Qcr1 and Qcr2 preassemble independently of complex III into matrix domain modules. Cryo-EM structures from a cardiolipin-deficient yeast strain and of recombinant protein resolved single, double and triple matrix domains, which are linked by the C-terminal tail of Qcr1. In the mature complex III, the same tail binds cytochrome b and cardiolipin. The modules form a dynamic pool, in which assembly species interconvert and exchange subunits. Mutations at the module interfaces, including mimics of human disease variants, reshape this pool and deletion of the Qcr1 tail impairs respiratory growth. Substitution of a tyrosine residue linked to parkinsonism exposes a conserved cysteine residue to oxidation. A cryo-EM structure of an assembly intermediate shows a cap of two matrix domains bound to a monomeric membrane module of cytochrome b, Qcr7, Qcr8 and cytochrome c1. We propose that complex III receives its matrix domain from a dynamic pool of prebuilt modules, which organizes the dimerization of the complex.

biochemistry↗
bioRxiv2026

Vasopressin V2 receptor phosphopeptide reshapes the conformational landscape of arrestin-2 beyond C-tail detachment

Binding of phosphorylated GPCR tails influences arrestin conformation, but the contribution of this interaction to the interdomain rotation associated with activation remains incompletely characterised. In particular, it remains unclear whether displacement of the arrestin C-tail is sufficient to favor larger interdomain rotations or whether the bound phosphotail exerts an additional effect on the conformational landscape. Here, we used enhanced-sampling simulations to characterise the conformational free-energy landscapes of basal (unbound) and V2Rpp-bound arrestin-2. We find that V2Rpp binding broadened the range of energetically accessible interdomain rotation angles relative to basal arrestin-2. However, the pronounced rotation observed in the experimental structures, while accessible, was not strongly favored. Strikingly, C-tail displacement alone does not reproduce this effect, as arrestin-2 retains a strong preference for small interdomain rotation angles in the absence of V2Rpp. Moreover, the C-tail-detached state is energetically unfavorable in the absence of V2Rpp. Together, these findings quantify the effects of V2Rpp binding on the interdomain rotation of arrestin-2 and disentangle the effects of phosphopeptide binding from those of C-tail detachment.

biochemistry↗
bioRxiv2026

Interventional breath analysis reveals oral microbial conversion of 1,2-Propanediol into Propanal and 1-Propanol

Background: Orally exhaled breath contains volatile organic compounds (VOCs) that may reflect local microbial metabolism as well as systemic processes. Consequently, the breath sampling route can influence VOC profiles and complicate biomarker interpretation. Previous studies have shown that 1-propanol is substantially enriched in orally compared with nasally exhaled breath. Objective: To determine whether a controlled oral challenge with 1,2-propanediol (1,2-PD) induces sampling route-dependent changes in breath 1-propanol concentrations. Methods: Ten healthy volunteers received 0.1 mL 1,2-PD diluted in approximately 50 mL water. The solution was used as an oral rinse for approximately 30 s and then expectorated. Matched oral and nasal end-tidal breath samples were collected before administration, immediately afterwards, and at 15-min intervals. Propanal, 1-propanol, acetol, acetone, and isoprene were analysed using gas chromatograph-ion mobility spectrometer (GC-IMS). Results: Oral 1,2-PD exposure produced a rapid and transient increase in exhaled propanal and 1-propanol signals, with the largest responses immediately after administration and at the second post-intervention measurement. In contrast, nasal signals remained comparatively stable. Acetone and isoprene showed no consistent intervention-related changes, regardless of the sampling route. No reproducible increase in acetol was detected, although interpretation was limited by analytical interference. Conclusion: A controlled oral 1,2-PD challenge elicited a route-specific propanal and 1-propanol response, consistent with local microbial metabolism of 1,2-PD within the oral cavity. Both fucose and 1,2-PD have been previously reported in human saliva, including elevated concentrations in selected disease-associated contexts. Bacteria can degrade salivary mucin glycans, releasing sugars including fucose. Together, these observations motivate investigation of a proposed pathway linking mucin-derived fucose to 1,2-PD and subsequent propanal and 1-propanol formation in the human oral cavity.

biochemistry↗