Exploring the Evolution of Ahl Lactonases

Alternatives to traditional bactericidal treatments have received increased attention in the wake of widespread microbial resistance. One potentially exploitable pathway, quorum sensing, is a process of bacterial communication in which small, diffusible molecules are used to coordinate gene expression on a population scale. Enzymatic degradation of these molecules has been shown to disrupt fundamental bacterial behaviors including, among others, biofilm formation. Identification of high-performance quorum-quenching enzymes is therefore relevant to food and medical industries, which are keen to manage the formation of these potentially hazardous microsurfaces.

Acyl homoserine lactones (AHLs) are commonly used by gram negative bacteria as signaling molecules in the quorum sensing pathway. They are enzymatically degraded by AHL lactonases, which are themselves a part of the larger metallo-beta-lactamase (MBL) family. We have utilized sequence similarity networks (SSNs) to select and systematically characterized over three dozen members of the AHL lactonase family, as well as a subset of evolutionarily related members outside of this group. Investigations were conducted into protein expression, thermostability, and activity against a range of different substrates. In an attempt to optimize these characteristics, particularly active enzymes were identified from the original pool, and ancestral reconstruction was used to infer their precursor sequences; these proteins were subsequently characterized as well.

Our results illuminate a clear story of protein evolution. Many AHL lactonases display weak activity outside of their native reactions, which connect them to neighboring subgroups within the larger MBL family. Within the AHL lactonase family specifically, many of the most active enzymes are grouped together when plotted on the same SSN, providing clear opportunities for further optimization. Some enzymes were identified with thermostabilities upwards of 95^oC, with ancestral sequences being more stable than their extant counterparts.

In total, our work speaks to the evolutionary trajectory of an important class of quorum quenching enzymes, and also provides the groundwork for subsequent forays into more detailed protein engineering.