Two types of mutants have been produced from a thiamine auxotroph of E.coli, 70-23. One (70-23-107) requires thiamine diphosphate (TDP) and the other (70-23-102) thiamine monophosphate (TMP) or TDP.Thiamine, TMP and TDP are all equally active for the parent, whereas thiamine is inactive for mutants, 70-23-102 and 70-23-107 ; TMP is inactive for mutant 70-23-107. The different growth responses between the parent and mutant organisms appear to be due to the second mutation which effects the phosphorylating steps of thiamine. Experimental results obtained with such mutant strains indicated that free thiamine is not involved in the de novo synthesis of TDP, but TMP, an exclusive product formed through the reaction between 2-methyl-4-amino-5-hydroxymethylpyrimidine pyrophosphate and 4-methyl-5-β-hydroxyethylthiazole monophosphate, is directly phosphorylated to form TDP. Exogenous thiamine, on the other hand, must be converted to TDP via the intermediate formation of TMP. Thus, thiamine pyrophosphokinase in E. coli, which was reported to exist in this organism, should be separated into two different enzymes, thiamine monophosphokinase and thiamine monophosphatekinase. It is suggested then that if the phosphorylation of thiamine were involved in the process of thiamine uptake, thiamine monophosphokinase would have play a role, because exogenous thiamine is first converted into TMP by this enzyme. Our experiments, however, revealed that the uptake of both TMP and TDP by mutant 70-23 cells were stimulated by glucose. Furthermore, the stimulatory effect of glucose was observed in the uptake of mutant 70-23-102 cells for thiamine and of mutant 70-23-107 cells for TMP. Since mutant 70-23-102 was lacking in the thiamine monophosphokinase activity and mutant 70-23-107 was the same in the thiamine monophosphatekinase activity, both enzymes might not be involved as an essential component in the uptake system. TMP and TDP are actually utilized by the mutant cells without dephosphorylation to thiamine.
View full abstract