Pyridoxine (PN) kinase catalyzes the phosphorylation of PN to pyridoxine 5′ phosphate (PNP), which is then oxidized to pyridoxal 5′-phosphate (PLP). PLP, which is the active form of vitamin B6, is an essential and ubiquitous coenzyme used by many enzymes involved in amino acid metabolism and by glycogen phosphorylase. The goal of this thesis is to identify the PN kinase gene in Escherichia coli K-12. First, we devised a genetic screen to isolate a miniTn10 (Cmr) insertion mutant lacking PN kinase activity by transposon random mutagenesis. Then, we cloned the PN kinase structural gene, designated pdxK, by complementation. DNA sequence analysis showed that pdxK was adjacent to, but opposite from, the crr sugar transport gene (53.95 min). P1 transduction and PCR mapping confirmed that the miniTn10(Cmr) insertion was in the pdxK reading frame. Growth properties of pdxK::miniTn10 (Cmr) mutants suggests that PN kinase, which also phosphorylates pyridoxal (PL) and pyridoxamine (PM) in vitro, functions solely in B6 vitamer salvage pathway and that E. coli contains at least one other PL kinase. The amino acid sequence of PdxK has signature motifs of the PfkB superfamily of carbohydrate kinase which includes phosphofructokinase and ribokinases. Blast search identified several PdxK homologs that might encode PN/PL/PM kinase in several other organisms

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Research Info

Identification of The Pdxk Gene That Encodes pyridoxine (Vitamin B6) Kinase in Escherichia Coli K-12