Threonine synthetase of Bacillus subtilis. The nature of an associated dehydratase activity.

The gene encoding threonine synthetase has been located by genetic transformation in Bacillus subtilis. Its position is adjacent and central to the genes encoding homoserine dehydrogenase and homoserine kinase. Its mutational sites are closely linked to, and interspersed among, several mutational sites affecting a minor but phenotypically significant threonine dehydratase (distinct from the major, biosynthetic threonine dehydratase of B. subtilis). This minor dehydratase activity differs from the biosynthetic threonine dehydratase in several important respects. (a) It accounts, when derepressed, for only 4% of the total threonine dehydratase activity of normal cells. (b) The genes encoding the two enzymes are separated by more than half of the chromosome. (c) It has a Michaelis constant for L-threonine nearly 10 times as large as that of the biosynthetic enzyme. (d) It is only mildly inhibited by L-isoleucine. (e) It requires phosphate ions for activity. Enzymatic studies with mutant strains show that both threonine synthetase and the minor dehydratase are concomitantly altered by single mutational events, while strains missing both activities regain both by reverse mutation at single gene frequency (lo-*). Furthermore, transformation studies utilizing sheared DNA at low concentrations show that single, indivisible mutational sites affect both activities. Finally, both enzyme activities, partially purified from a strain derepressed for the enzymes of threonine production, co-fractionate to over 200-fold purification through standard protein purification procedures. These findings, together with the mutant studies, are an indication that the minor threonine dehydratase of B. subtifis is a secondary activity of threonine synthetase.