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

Lysine 2-hydroxyisobutyrylation (K) is a novel protein posttranslational modification conserved in eukaryotes and prokaryotes. However, the biological significance of K remains largely unknown. Here, through screening the proteome-wide K modification sites in bacteria using a bioinformatic method, we identified a potential K site (K201) 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 of FabI in an in vitro experiment. To investigate the biological effects of the K 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 reduced FabI activity with decreased Michaelis constant (K) and catalytic turnover number (k), while the mutant mimicking the unmodified form and the recombinant wild-type protein treated with CobB exhibited increased activity. However, the dissociation constant (K) between FabI and NADH was not affected by the mutation mimicking the modification, suggesting that K201 didn't alter the binding between NADH and FabI. We also found that K201 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 on FabI activity and pointed to a novel mechanism related to antibiotic resistance.