Role of pyridoxal kinase in vitamin B6 uptake by Escherichia coli.

Abstract

E. coli KG980, a vitamin B6 auxotroph derived from wild strain K12, concentrated exogenous pyridoxal in an energy-dependent manner, and the effects of energy sources and inhibitors on pyridoxal uptake compared with those on proline uptake indicated that the energy required was in the form of phosphate bonds and not of membrane potential. The vitamin taken up was primarily present as pyridoxal 5''-phosphate and pyrodoxamine 5''-phosphate intracellularly, and energy depletion decreased the accumulation as the phosphorylated derivatives but not as unaltered pyridoxal itself. The intracellular phosphorylation, which requires ATP, was essential for the concentrative uptake of the vitamin. Pyridoxal oxime inhibited pyridoxal uptake by decreasing the intracellular phosphorylation without affecting the entry of pyridoxal across the cell membrane. A pyridoxal kinase deficient mutant (HN1) derived from the strain KG980 showed a low ability to take up pyridoxal because of the failure to accumulate it effectively as phosphorylated derivatives. The carrier-mediated nature of pyridoxal uptake, previously suggested by saturation kinetics, was further supported by the finding that 4''-deoxypyridixine inhibited pyridoxal uptake competitively, decreasing the intracellular appearance of unmetabolized pyridoxal. Pyridoxal may enter the cells by facilitated diffusion. Pyridoxal is probably accumulated by conversion to phosphorylated derivatives. Similar results on the uptake of pyridoxine and pyridoxamine are also presented.