The Thiamine Kinase (YcfN) Enzyme Plays a Minor but Significant Role in Cobinamide Salvaging in Salmonella enterica

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	Abstract

Cobinamide (Cbi) salvaging is impaired, but not abolished, in a Salmonella enterica strain lacking a functional cobU gene. CobU is a bifunctional enzyme (NTP:AdoCbi kinase, GTP:AdoCbi-P guanylyltransferase) whose AdoCbi kinase activity is necessary for Cbi salvaging in this bacterium. Inactivation of the ycfN gene in a cobU strain abrogated Cbi salvaging. Introduction of a plasmid carrying the ycfN⁺ allele into a cobU ycfN strain substantially restored Cbi salvaging. Mass spectrometry data indicate that when YcfN-enriched cell-free extracts were incubated with AdoCbi and ATP, the product of the reaction was AdoCbi-P. Results from bioassays confirmed that YcfN converted AdoCbi to AdoCbi-P in an ATP-dependent manner. YcfN is a good example of enzymes that are used by the cell in multiple pathways to ensure the salvaging of valuable precursors.