Activity Improvement and Vital Amino Acid Identification on the Marine-Derived Quorum Quenching Enzyme MomL by Protein Engineering

Abstract

MomL is a marine-derived quorum-quenching (QQ) lactonase which can degrade various N-acyl homoserine lactones (AHLs). Intentional modification of MomL may lead to a highly efficient QQ enzyme with broad application potential. In this study, we used a rapid and efficient method combining error-prone polymerase chain reaction (epPCR), high-throughput screening and site-directed mutagenesis to identify highly active MomL mutants. In this way, we obtained two candidate mutants, MomLI144V and MomLV149A. These two mutants exhibited enhanced activities and blocked the production of pathogenic factors of Pectobacterium carotovorum subsp. carotovorum (Pcc). Besides, seven amino acids which are vital for MomL enzyme activity were identified. Substitutions of these amino acids (E238G/K205E/L254R) in MomL led to almost complete loss of its QQ activity. We then tested the effect of MomL and its mutants on Pcc-infected Chinese cabbage. The results indicated that MomL and its mutants (MomLL254R, MomLI144V, MomLV149A) significantly decreased the pathogenicity of Pcc. This study provides an efficient method for QQ enzyme modification and gives us new clues for further investigation on the catalytic mechanism of QQ lactonase.

Prevention of biofilm formation by quorum quenching

“…Some bacterial species such as Pseudomonas aeruginosa, Klebsiella pneumoniae, Bacillus spp., and Agrobacterium tumefaciens produce enzymes that are capable of degrading AHL molecules. MomL lactonase isolated from Muricauda olearia Th120 has the ability to degrade both long-and shortchain AHL equally, thus inhibiting virulence in many pathogenic bacteria (Wang et al 2019a).In addition, the ability to produce enzymes that degrade AHL molecules and thereby interfere with QS has also been found in plants and fungi such as Pachyrhizus erosus, Lotus corniculatus, and Hordeum vulgare. Medicinal plants produce a wide spectrum of secondary metabolites such as flavonoids, phenols, phenolic acids, saponins, coumarins, tannins, quinones, terpenoids, alkaloids, and polyacetylenes, against the QS system.…”

Engineering acyl-homoserine lactone-interfering enzymes toward bacterial control

“…Indeed, this strain is impaired (by transposon insertions) in AHL synthesis (40), but its endogenous QS system is functional, and QS activation leads to the production of the purple pigment violacein, which acts as an antibiotic at high cell density. Therefore, it can be used for screening purposes and responds best to exogenous AHLs with chain lengths ranging from C4-HSL to C8-HSL, including OC6-HSL and OC8-HSL (41)(42)(43). In particular, C. violaceum CV026 was used to screen a metagenomic library of 250,000 clones from a hypersaline soil located in Spain with a pool strategy of 50 clones/well, which resulted in the identification of a single lactonase, called HqiA, which had no homology with any known lactonase or acylase.…”

“…The hydrolysis of one lactone molecule leads to the generation of one proton, resulting in the acidification of the medium. Consequently, enzyme kinetics can be monitored with a pH indicator molecule, such as cresol purple or bromothymol blue, in a colorimetric assay (43,48,49). This assay can be miniaturized and performed in microtiter plates, but its high background levels make it challenging to use with cell lysates.…”

“…Similarly, the variants MomL I144V and MomL V149A obtained by random engineering were able to decrease infection of Chinese cabbage by Pcc. Cabbage leaves have been infected with Pcc, in the presence of MomL variants or controls, on a cut surface of the leaf; thus, MomL variants greatly reduced decay areas (43).…”

Engineered Bacteriophages for Practical Applications

“…the use of a quorum-quenching enzyme to disrupt bacterial communication, leading to the inhibition of biofilm formation. 17) With the number of phages already sequenced, and the techniques available to improve enzyme function [18][19][20] and phage manipulation, these approaches could constitute a useful weapon to incorporate into future strategies.…”