Catalytic Redundancies and Conformational Plasticity Drives Selectivity and Promiscuity in Quorum Quenching Lactonases.

Several enzymes from the metallo-ß-lactamase-like family of lactonases (MLLs) degrade N- acyl-L-homoserine lactones (AHLs). In doing so, they play a role in a microbial communication system, quorum sensing, which contributes to pathogenicity and biofilm formation. There is currently great interest in designing quorum quenching ( QQ ) enzymes that can interfere with this communication and be used in a range of industrial and biomedical applications. However, tailoring these enzymes for specific targets requires a thorough understanding of their mechanisms and the physicochemical properties that determine their substrate specificities. We present here a detailed biochemical, computational, and structural study of the MLL GcL, which is highly proficient, thermostable, and has broad substrate specificity. Strikingly, we show that GcL does not only accept a broad range of substrates but is also capable of utilizing different reaction mechanisms that are differentially used in function of the substrate structure or the remodeling of the active site via mutations. Comparison of GcL to other lactonases such as AiiA and AaL demonstrates similar mechanistic promiscuity, suggesting this is a shared feature across lactonases in this enzyme family. Mechanistic promiscuity has previously been observed in the lactonase/paraoxonase PON1, as well as with protein tyrosine phosphatases that operate via a dual general-acid mechanism. The apparent prevalence of this phenomenon is significant from both a biochemical and an engineering perspective: in addition to optimizing for specific substrates, it is possible to optimize for specific mechanisms, opening new doors not just for the design of novel quorum quenching enzymes, but also of other mechanistically promiscuous enzymes.

Engineering quorum quenching acylases with improved kinetic and biochemical properties.

Many Gram-negative bacteria use N-acyl-L-homoserine lactone (AHL) signals to coordinate phenotypes such as biofilm formation and virulence factor production. Quorum-quenching enzymes, such as AHL acylases, chemically degrade these molecules which prevents signal reception by bacteria and inhibits undesirable biofilm-related traits. These capabilities make acylases appealing candidates for controlling microbes, yet candidates with high activity levels and substrate specificity 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 generate these improved properties using the Protein One-Stop Shop Server. The engineering of acylases is complicated by low-throughput enzymatic assays. Alleviating this challenge, we report a time-course kinetic assay for AHL acylases that monitors the real-time production of homoserine lactone. Using the assay, we identified variants of PvdQ that were significantly stabilized, with melting point increases of up to 13.2°C, which translated into high resistance against organic solvents and increased compatibility with material coatings. While the MacQ mutants were unexpectedly destabilized, they had considerably improved kinetic properties, with >10-fold increases against N-butyryl-L-homoserine lactone and N-hexanoyl-L-homoserine lactone. 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.

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 °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.

Identification and biochemical characterization of a heteromeric cis-prenyltransferase from the thermophilic archaeon Archaeoglobus fulgidus.

cis-Prenyltransferases (cPTs) form linear polyprenyl pyrophosphates, the precursors of polyprenyl or dolichyl phosphates that are essential for cell function in all living organisms. Polyprenyl phosphate serves as a sugar carrier for peptidoglycan cell wall synthesis in bacteria, a role that dolichyl phosphate performs analogously for protein glycosylation in eukaryotes and archaea. Bacterial cPTs are characterized by their homodimeric structure, while cPTs from eukaryotes usually require two distantly homologous subunits for enzymatic activity. This study identifies the subunits of heteromeric cPT, Af1219 and Af0707, from a thermophilic sulphur-reducing archaeon, Archaeoglobus fulgidus. Both subunits are indispensable for cPT activity, and their protein-protein interactions were demonstrated by a pulldown assay. Gel filtration chromatography and chemical cross-linking experiments suggest that Af1219 and Af0707 likely form a heterotetramer complex. Although this expected subunit composition agrees with a reported heterotetrameric structure of human hCIT/NgBR cPT complex, the similarity of the quaternary structures is likely a result of convergent evolution.

A heteromeric cis-prenyltransferase is responsible for the biosynthesis of glycosyl carrier lipids in Methanosarcina mazei.

Cis-prenyltransferases are enzymes responsible for the biosynthesis of glycosyl carrier lipids, natural rubber, and some secondary metabolites. Certain organisms, including some archaeal species, possess multiple genes encoding cis-prenyltransferase homologs, and the physiological roles of these seemingly-redundant genes are often obscure. Cis-prenyltransferases usually form homomeric complexes, but recent reports have demonstrated that certain eukaryotic enzymes are heteromeric protein complexes consisting of two homologous subunits. In this study, three cis-prenyltransferase homolog proteins, MM_0014, MM_0618, and MM_1083, from the methanogenic archaeon Methanosarcina mazei are overexpressed in Escherichia coli and partially purified for functional characterization. Coexistence of MM_0618 and MM_1083 exhibits prenyltransferase activity, while each of them alone has almost no activity. The chain-lengths of the products of this heteromeric enzyme are in good agreement with those of glycosyl carrier lipids extracted from M. mazei, which are likely di- and tetra-hydrogenated decaprenyl phosphates, suggesting that the MM_0618/MM_1083 heteromer is involved in glycosyl carrier lipid biosynthesis. MM_0014 acts as a typical homomeric cis-prenyltransferase and produces shorter products.