The microbial enzyme 5’-methylthioadenosine/S-adenosylhomocysteine (MTA/SAH) nucleosidase (MTN) has three substrates MTA, SAH and 5’-deoxyadenosine (5’dADO). In each case, MTN cleaves the glycosidic linkage between the adenine ring and ribose sugar. MTN plays an essential part of methionine and purine salvage pathways, the activated methyl cycle, autoinducer-2 production, and radical S-adenosylmethionine (SAM) dependent reactions that are integral to vitamin synthesis. In this study, the impact of MTN activity on vitamin production and vitamin dependent metabolism were studied in <i>E. coli</i> strain O157:H7 wild type (WT) and MTN knock-out (KO) cells. Vitamins serve as cofactors for multiple pathways involved in bacterial metabolism. Proteomic studies comparing WT and KO strains have shown that the MTN KO strain expresses altered levels of enzymes involved in vitamin synthesis (thiamine, lipoate, biotin, etc.) and downstream enzymes that depend on these vitamins for activity. The results of our studies show that the activity of the biotin-containing enzyme acetyl-coA carboxylase (ACC) is reduced in the MTN KO strain. ACC is required for fatty acid biosynthesis. By biotin ELISA and western blot, we see a dramatic decrease in native biotinylation of ACC. Similarly, the activity of thiamine and lipoate dependent enzymes were also found to be reduced. We propose that loss of MTN activity leads to an accumulation of 5’dADO that in turn leads to product inhibition of radical SAM reactions, thus altering global metabolism through decreases in activity of vitamin dependent enzyme steps. Since MTN is only present in microbes, not humans, and central to metabolism, it may be a good target for antibiotic development. Our studies are useful in demonstrating potential mechanisms of action for such antibiotics.

MTN knockout attenuates vitamin synthesis and global metabolism in <i>E. coli</i> O157:H7

2019 Spring National Meeting

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MTN knockout attenuates vitamin synthesis and global metabolism in E. coli O157:H7