Abstract
Two types of mutants of Escherichia coli were isolated, one of which (mutant 70-23-107) responded to thiamine pyrophosphate, and the other (mutant 70-23-102) to thiamine monophosphate and thiamine pyrophosphate. They were produced by further mutation of a thiamine auxotroph of E. coli 70-23 with N-methyl-N′-nitro-N-nitrosoguanidine. The parent organism required thiamine because phosphohydroxymethylpyrimidine kinase activity was lacking in this organism, and hydroxymethylpyrimidine pyrophosphate was not permeable through the cell membrane of E. coli. Thiamine, thiamine monophosphate, and thiamine pyrophosphate were all equally active for the parent, whereas mutants 70-23-102 and 70-23-107 lost their ability to grow on thiamine. Both mutants differed only in the growth response to thiamine monophosphate: the former could grow on thiamine monophosphate, whereas the latter could not. Experimental results with the newly isolated mutants indicate that in E. coli the free form of thiamine is not involved in de novo synthesis of thiamine pyrophosphate, but thiamine monophosphate, an exclusive product formed by the reaction between hydroxymethylpyrimidine pyrophosphate and hydroxyethylthiazole monophosphate, is directly phosphorylated to form thiamine pyrophosphate. Exogenous thiamine, on the other hand, is converted to thiamine pyrophosphate via the intermediate formation of thiamine monophosphate.
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