A New Intact Cell System for Determination of the Rate of Vitamin B6 Biosynthesis

概要

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To establish an intact cell system to determine the rate of vitamin B6 (B6) biosynthesis, conditions suitable for the reaction were examined. Cells of Escherichia coli B WG2, a pdxH. mutant, were used for the reaction. Pyridoxal-starved cells of a stationary phase culture showed a negligible change of cell mass and a constant increase of B6 in the reaction. The rate of B6 biosynthesis was reliably determined with a 2-hr reaction initiated with cells of 0.10 to 0.15mg per ml. The influence of endogenous metabolites was completely abolished by the use of such a low concentration of the starved cells. When glucose was used as a sole carbon substrate in the reaction system, the maximum rate of B6 biosynthesis was determined to be 0.5 to 0.6 nmol per mg cells per hr. The maximum rate increased to 0.8 to 0.9 nmol per mg cells per hr on supplementation of glycolaldehyde to the reaction mixture. The result led to the conclusion that the rate-limiting step in the reaction sequence of B6 biosynthesis lies the process of incorporation of the 5-5 carbon fragment of the B6 molecule. This was also confirmed by a growth experiment with the wild type strain.
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