Studies on the biosynthesis of vitamin B6 and the pyrimidine of vitamin B1 in Saccharomyces cerevisiae

Thiamin, also known as vitamin B1, is an essential cofactor in all living systems. Despite being one of the first vitamins discovered, many aspects of thiamin biosynthesis remain to be elucidated. Previous labeling studies in S. cerevisiae have shown that the 4-amino-2-methyl-5-hydroxymethylpyrimidine (HMP) moiety of thiamin is derived from histidine and pyridoxine (vitamin B6) and genetic knockout results demonstrated that the THI5 gene family is involved in HMP production. Interestingly, in the yeast genome THI5 is located adjacent to SNO and SNZ, putative pyridoxal-5'-phosphate (PLP) biosynthetic genes, and these genes are known to be regulated by thiamin availability. The incorporation of pyridoxine (a PLP vitamer) into HMP suggested a possible role for SNO and SNZ in HMP biosynthesis. The investigations described in this work aimed to elucidate the components needed for HMP biosynthesis in S. cerevisiae and reconstitute HMP biosynthetic activity in vitro.; The overexpression and biochemical characterization of SNOp and SNZp confirmed that they are indeed PLP biosynthetic enzymes. Complementation experiments in an E. coli HMP auxotroph established that THI5 is capable of producing HMP, and that this activity does not require additional eukaryotic genes. The first in vitro reconstitution of the HMP biosynthetic activity of purified THI5p is described. THI5p is oxygen sensitive, requires both oxygen and one equivalent of iron for catalysis, and utilizes PLP as a substrate. PLP is also shown to inhibit HMP production by THI5p if it is present at stoichiometries higher than 1 to 1. THI5p appears to prefer SNZ3p-bound PLP compared to free PLP, which may indicate a role for SNZ3p in delivering PLP to THI5p in ratios that maximize HMP production. The role of histidine in the THI5p reaction is explored but remains unclear.