Engineering Quorum Quenching Acylases with Improved Kinetic and Biochemical Properties

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