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

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.
Competing Interests: Declaration of competing interest Authors have no competing interests to declare.
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