The Biosynthesis of Thiamine

Abstract

In Paper I of this series (1) the enzymatic formation of phosphate esters of 2-methyl-4-amino-5-hydroxymethylpyrimidine was demonstrated and evidence was presented that in bakers' yeast pyrimidine-pyrophosphate is an intermediate in the biosynthesis of thiamine.In the present paper, data are presented to show that pyrimidine-PP reacts with the monophosphate ester of 4-methyl-5-(P-hydroxyethyl)thiazole to yield thiamine-P as the initial thiamine compound which is formed enzymatically.Data are also presented which indicate that thiamine-P thus formed must first be dephosphorylated to thiamine before it can be converted enzymatically to thiamine-PP, the coenzyme form of thiamine.Details of the fractionation of the enzyme system responsible for the biosynthesis of thiamine and its phosphate esters are also presented.EXPERIMENTAL PROCEDURE Materials-ATP was obtained from Pabst, Inc. Generous amounts of 2-methyl-4-amino-5-hydroxymethylpyrimidine and 4-methyl-5-(&hydroxyethyl)thiazole were kindly supplied by Merck and Company, Inc. DEAE-cellulose1 was a commercial product obtained from Eastman Organic Chemicals.Alkaline phosphatase was a gift from Drs. F. Rothman and C. Levinthal.Pyrimidine-PP was produced enzymatically and freed from ATP and pyrimidine-P by the methods described in Paper I of this series (1).Thiamine-P was purchased from Nutritional Biochemicals Company; thiamine-PP was a product of Merck and Company, Inc. Treatment of DEAE-Cellulose Used for Chromatography-A suspension of 18 g of DEAE-cellulose in 500 ml of 0.1 M EDTA (adjusted to pH 10 with NaOH) was stirred for 20 minutes, filtered through glass wool, and the resin washed with 1500 ml of distilled water.The resin then was resuspended in 500 ml of NaOH, stirred for 15 minutes, and again filtered through glass