Abstract

Members of the LuxI protein family catalyze synthesis of acyl-homoserine lactone (acyl-HSL) quorum sensing signals from S-adenosyl-L-methionine and an acyl thioester. Some LuxI family members prefer acyl-CoA, and others prefer acyl-acyl carrier protein (ACP) as the acyl-thioester substrate. We sought to understand the evolutionary history and mechanisms mediating this substrate preference. Our phylogenetic and motif analysis of the LuxI acyl-HSL synthase family indicates that the acyl-CoA-utilizing enzymes evolved from an acyl-ACP-utilizing ancestor. To further understand how acyl-ACPs and acyl-CoAs are recognized by acyl-HSL synthases we studied BmaI1, an octanoyl-ACP-dependent LuxI family member from Burkholderia mallei, and BjaI, an isovaleryl-CoA-dependent LuxI family member from Bradyrhizobium japonicum. We synthesized thioether analogs of their thioester acyl-substrates to probe recognition of the acyl-phosphopantetheine moiety common to both acyl-ACP and acyl-CoA substrates. The kinetics of catalysis and inhibition of these enzymes indicate that they recognize the acyl-phosphopantetheine moiety and they recognize non-preferred substrates with this moiety. We find that CoA substrate utilization arose through exaptation of acyl-phosphopantetheine recognition in this enzyme family.

Highlights

  • Bacterial quorum sensing is a genetic regulatory phenomenon whereby cells excrete or secrete a chemical signal into the surrounding environment and at sufficient concentrations the signal alters expression of specific genes [1,2,3]

  • Knowledge of acyl-HSL quorum sensing has been applied in many synthetic biology studies [4], and different strategies to evolve acyl-HSL synthases have been employed [5,6,7]

  • Our work indicates known acyl-CoA-utilizing acyl-HSL synthases evolved from an ancestral acyl-acyl carrier protein (ACP)-utilizing enzyme through application of acyl-PPant recognition to acyl-CoA substrates

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Summary

Introduction

Bacterial quorum sensing is a genetic regulatory phenomenon whereby cells excrete or secrete a chemical signal into the surrounding environment and at sufficient concentrations the signal alters expression of specific genes [1,2,3]. Many Proteobacteria use acyl-homoserine lactones (acyl-HSLs) as quorum sensing signals. Knowledge of acyl-HSL quorum sensing has been applied in many synthetic biology studies [4], and different strategies to evolve acyl-HSL synthases have been employed [5,6,7]. Because acyl-HSL quorum sensing affects the virulence of some bacterial pathogens, there have been many efforts to identify inhibitors of acyl-HSL receptor proteins, acyl-HSL synthases, or both [8,9,10,11]. The crystal structures of three acyl-ACP-dependent acyl-HSL synthases have been solved [10,18,19], and it is apparent from the structures that these three enzymes are part of the Gcn N-

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