The natural occurrence and enzymatic formation of a hydroxyethyl derivative of thiamine pyrophosphate.

Breslow (1) has formulated a mechanism of thiamine action in which the Z-position of the thiazolium ring of thiamine is postulated to be the reactive part of the molecule. Krampitz et al. (2) have prepared thiamine with an cr-hydroxyethyl group substituted on the Z-position and reported its probable identity as “active acetaldehyde.” Recent results from thii laboratory indicate that the pyrophosphoric acid ester of ar-hydroxyethyl thiamine1 occurs naturally in microorganisms. Acid extracts of Escherichiu coli cells, after dephosphorylation by treatment with Taka-Diastase, contained a compound which migrated on paper chromatograms identically with synthetic ar-hydroxyethyl thiamine2 in six different solvent systems (Table I). Zones of migration were determined by the bioautographic technique (3) with Lactobmillus viridescens as the test organism. The relative sizes of the growth zones indicated that 60 to 75% and about 25% of the total thiamine present in E. coli and bakers’ yeast, respectively, could be accounted for as phosphorylated cr-hydroxyethyl thiamine. Other evidence which indicates that the dephosphorylated product is identical with synthetic cr-hydroxyethyl thiamine consists of the observations that both the natural and synthetic compounds behave in a similar manner in replacing thiamine as a growth factor for microorganisms. Thus, both can be utilized efficiently by L. viriofescens, Lactobacillus fermenti, and Kloeckera brevis, but neither can be utilized by a thiamine-requiring mutant of E. coli. It has been possible to form, enzymatically, the pyrophosphate ester of a-hydroxyethyl thiamine by incubating thiamine pyrophosphate with wheat germ carboxylase (purified to the alcohol powder stage (4)) and either pyruvate or acetaldehyde. After incubation, treatment of the reaction mixtures with Taka-Diastase followed by chromatography and bioautography revealed that 10 to 12% of the thiamine pyrophosphate had been con-