Biochemical and Structural Basis of Triclosan Resistance in a Novel Enoyl-Acyl Carrier Protein Reductase [PublishAheadOfPrint]

Enoyl-acyl carrier protein reductase (ENR), such as FabI, FabL, FabK and FabV, catalyzes the last reduction step in bacterial type II fatty acid biosynthesis. Previously, we reported metagenome-derived ENR homologs resistant to triclosan (TCL) and highly similar to 7-α hydroxysteroid dehydrogenase (7-AHSDH). These homologs are commonly found in Epsilonproteobacteria, a class that contains several human pathogenic bacteria, including the genera Helicobacter and Campylobacter. Herein, we report the biochemical and predicted structural basis of TCL resistance in a novel 7-AHSDH-like ENR. The purified protein exhibited NADPH-dependent ENR activity but no 7-AHSDH activity, despite its high homology with 7-AHSDH (69%-96%). Because this ENR was similar to FabL (41%), we propose that this metagenome-derived ENR is referred to as FabL2. Homology modeling, molecular docking, and molecular dynamic simulation analyses revealed the presence of an extrapolated six-amino acid loop specific to FabL2 ENR, which prevented the entry of TCL into the active site of FabL2 and was likely responsible for TCL resistance. Elimination of this extrapolated loop via site-directed mutagenesis resulted in the complete loss of TCL resistance but not enzyme activity. Phylogenetic analysis suggested that FabL, FabL2, and 7-AHSDH diverged from a common short-chain dehydrogenase reductase family. This study is the first to report the role of the extrapolated loop of FabL2-type ENRs in conferring TCL resistance. Thus, the FabL2 ENR represents a new drug target specific for pathogenic bacteria of Epsilonproteobacteria.

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