Effects of lysine 2-hydroxyisobutyrylation on bacterial FabI activity and resistance to triclosan

Abstract

Lysine 2-hydroxyisobutyrylation (K_hib) is a novel protein posttranslational modification conserved in eukaryotes and prokaryotes. However, the biological significance of K_hib remains largely unknown. Here, through screening the proteome-wide K_hib modification sites in bacteria using a bioinformatic method, we identified a potential K_hib site (K201_hib) targeted by de-2-hyroxyisobutyrylase CobB at the substrate-binding site of FabI, an enoyl-acyl carry protein reductase (EnvM or FabI) in fatty acid biosynthesis pathway. First, we confirmed that the previously identified de-2-hyroxyisobutyrylase CobB can remove K_hib of FabI in an in vitro experiment. To investigate the biological effects of the K_hib on FabI's activity, amino acid substitutes were introduced to the modification sites of the protein of E. coli origin to mimic modified/unmodified status. We found that the mutant mimicking K201_hib reduced FabI activity with decreased Michaelis constant (K_m) and catalytic turnover number (k_cat), while the mutant mimicking the unmodified form and the recombinant wild-type protein treated with CobB exhibited increased activity. However, the dissociation constant (K_D) between FabI and NADH was not affected by the mutation mimicking the modification, suggesting that K201_hib didn't alter the binding between NADH and FabI. We also found that K201_hib tended to increase the resistance of E. coli to triclosan (TCL), a widely-used antibiotics targeting FabI. Taken together, this study identified the regulatory role of K_hib on FabI activity and pointed to a novel mechanism related to antibiotic resistance.

Keywords: Antibiotic resistance; FabI; Lysine 2-hydroxyisobutyrylation; Posttranslational modification; Triclosan.