A large number of enzymes remain uncharacterized even in Escherichia coli (E.coli) K-12 whose genome has been known for over 15 years. Metabolomics can be useful to study such enzymes and particularly to identify their endogenous substrates. Here, we combined classical enzyme assay and metabolite profiling using Capillary electrophoresis-mass spectrometry (CE-MS) to characterize the activity of YhhY, a putative metal-inducible acetyltransferase. Screening using CoA assays revealed that the enzyme could transfer acetyl groups to the N-terminal of amino acids, preferably methionine, phenylalanine and histidine. The enzyme was found to be inhibited by thiol-reactive agents and kinetics date shows YhhY follows a ternary complex mechanism, and reaction rates differ among each amino acid.
To confirm the in vitro results and rule out the possibility of activity resulting from a contaminating enzyme, we monitored metabolite changes by CE-MS in E.coli cells that overexpress YhhY. We observed elevated levels of acetylated methionine, phenylalanine and histidine in the overexpressing cells, confirming that the amino acid acetylating activity is connected with YhhY. Finally, in order to better understand the physiological activity of YhhY, we profiled metabolites in E.coli cells in which the yhhY gene has been disrupted. Following treatment with cobalt, we observed broad metabolite changes between wild type and knock out strain. The most significant changes were in nucleotide, pentose phosphate, and glutathione pathway intermediates. Overall, our results demonstrate that YhhY is an acetyltransferase displaying activity on several amino acids.
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