More than 60 years after the discovery and synthesis of pyridoxine (vitamin B6) and of thiamin (vitamin B1) and the recognition of their nutritional importance, and more than 30 years after the elucidation of their biochemical functions, knowledge of the biosynthetic pathways leading to each compound is still fragmentary. The objectiveof the present proposal is the further elucidation of the biosynthesis of the two vitamins and, more specifically, the search for committed precursors on the metabolic route to each of them. Based on the results of incorporation studies with radioactive tracers, which showed that the carbon chains of each of the two vitamins are derived from carbohydrate precursors, working hypotheses have been put forward that account for the mechanism of biosynthesis of pyridoxine and of each of the two subunits of thiamin and that postulate structures of advanced intermediates. In the present grant period it is intended to test these proposals. 1-Deoxy-D-xylulose and 4-hydroxy-L-threonine will be synthesized and tested as precursors of pyridoxine in E. coli. Three approaches will be employed which, if successful, would provide compelling evidence in support of the intermediacy of these two compounds: (1) displacement of incorporation of label from (6-13C)glucose in the presence of unlabeled samples of 1-deoxy-D-xylulose and 4-hydroxy-L-threonine; (2) incorporation of label from 13C-labeled samples of 1-deoxy-D-xylulose and 4-hydroxy-L-threonine; and (3) trapping of 3H/14C-labeled 1-deoxy-D-xylulose and 4-hydroxy-L-threonine from an E. coli culture that has yielded 3H/14C-labeled pyridoxine from 3H/14C-labeled glycerol. If 1-deoxy-D-xylulose is confirmed as an intermediate, its oxidationproduct, 2,4-dioxopentane-3,5-diol, will be tested similarly. Incorporation of 13C-labeled 1-deoxy-D-xylulose into the C5 chain of the thiazole nucleus of thiamin and of specifically 13C-labeled hexoses into the pyrimidine nucleus will be tested in an attempt to provide answers to outstanding questions of their origin. It is becoming evident that the primary precursors of B1 and B6 are common metabolites. It is the long term objectiveof this investigation to identify the step in each pathway that is blocked in mammalian tissues, thereby making the compounds essential dietary components.
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