Search bioRxiv⌕ Search
Home/Archive/bioRxiv

bioRxiv · 10.1101/2023.09.01.555929

Engineering Quorum Quenching Acylases with Improved Kinetic and Biochemical Properties

Sompiyachoke, K.·Elias, M.
View original source ↗Export metadata ↓

Abstract

Many Gram-negative bacteria respond to N-acyl-L-homoserine lactone (AHL) signals to coordinate phenotypes such as biofilm formation and virulence factor production. Quorum-quenching enzymes, such as acylases, chemically degrade AHL signals, prevent signal reception by bacteria, and inhibit undesirable traits related to biofilm. These capabilities make these enzymes appealing candidates for controlling microbes. Yet, enzyme candidates with high activity levels, high substrate specificity for specific interference, and that are capable of being formulated into materials are needed. In this work, we undertook engineering efforts against two AHL acylases, PvdQ and MacQ, to obtain improved acylase variants. The engineering of acylase is complicated by low-throughput enzymatic assays. To alleviate this challenge, we report a time-course kinetic assay for AHL acylase that tracks the real-time production of homoserine lactone. Using the protein one-stop shop server (PROSS), we identified variants of PvdQ that were significantly stabilized, with melting point increases of up to 13.2 {degrees}C, which translated into high resistance against organic solvents and increased compatibility with material coatings. We also generated mutants of MacQ with considerably improved kinetic properties, with >10-fold increases against N-butyryl-L-homoserine lactone and N-hexanoyl-L-homoserine lactone. In fact, the variants presented here exhibit unique combinations of stability and activity levels. Accordingly, these changes resulted in increased quenching abilities using a biosensor model and greater inhibition of virulence factor production of Pseudomonas aeruginosa PA14. While the crystal structure of one of the MacQ variants, M1, did not reveal obvious structural determinants explaining the observed changes in kinetics, it allowed for the capture of an acyl-enzyme intermediate that confirms a previously hypothesized catalytic mechanism of AHL acylases. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=69 SRC="FIGDIR/small/555929v1_ufig1.gif" ALT="Figure 1"> View larger version (31K): org.highwire.dtl.DTLVardef@12393cdorg.highwire.dtl.DTLVardef@bc8cd4org.highwire.dtl.DTLVardef@c1e2e3org.highwire.dtl.DTLVardef@116bc09_HPS_FORMAT_FIGEXP M_FIG C_FIG

Source connections

Explore related subjects

biochemistryPreprint

Keep this discovery

Explore connections, maps & timelines

BibTeXRIS

Sompiyachoke, K., Elias, M.. 2023-09-01. Engineering Quorum Quenching Acylases with Improved Kinetic and Biochemical Properties. https://doi.org/10.1101/2023.09.01.555929

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

KEEP EXPLORING

Related preprints

bioRxiv2026

EngA adopts multiple distinct conformational states during ribosome assembly

Ribosome assembly, a highly coordinated and crucial cellular process, is assisted by various assembly factors, including GTPases. EngA, an essential P-loop GTPase, is involved in late-stage assembly of the 50S ribosomal subunit. The structures of 50S ribosome in complex with EngA-bound GMPPNP, and EngA-bound GDP alone, without 50S subunit, have been reported previously. However, the molecular mechanism of EngA GTP hydrolysis-assisted 50S maturation remains poorly understood, and structures of the 50S ribosome in complex with EngA-bound GDP have yet to be deciphered. We have reported cryo- EM structures of the Mycobacterium smegmatis 50S ribosome in complex with EngA-bound GTP{gamma}S or GDP at 2.8 - 3.5 [A] resolution. Structural analysis revealed that EngA and 50S subunit undergo a coordinated structural rearrangement, and an assembly intermediate is formed in which the stem-loop of H80 makes base-pair interactions with the junction of H89 and H90, forming a unique stem-loop-junction (SLJ) motif. EngA-bound GDP adopts distinctly different conformations in its ribosome-bound and unbound states. In summary, this study proposed a multi-state conformational model for EngA-assisted ribosome assembly.

biochemistry↗
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↗