In vivo and enzymatic conversion of toyocamycin to sangivamycin by Streptomyces rimosus

The pyrrolopyrimidine nucleosides, toyocamycin, sangivamycin, and tubercidin are isolated from the culture filtrates of 14 species of the Streptomyces. Although earlier experiments showed that the biosynthesis of the pyrrolopyrimidine nucleosides require GTP as the common precursor, there was no experimental evidence to demonstrate the interconversion of these naturally occurring nucleoside analogs. The data presented here describe two types of experiments to prove that toyocamycin is the precursor for sangivamycin. First, in vivo experiments show that radioactive toyocamycin is converted to sangivamycin. Second, the enzyme, toyocamycin nitrile hydrolase, that catalyzes the conversion of toyocamycin to sangivamycin has been isolated and partially purified from the soluble fraction of S. rimosus. The nitrile hydrolase is not present in cell-free extracts of the Streptomyces that synthesize tubercidin or toyocamycin. Activity can be assayed by measuring the formation of radioactive sangivamycin from toyocamycin. The enzyme has been purified 24-fold with an over-all yield of 5%. The pH optimum is 6.5 and the K_m is 0.5 mm. Most nitriles tested are competitive inhibitors but they are not substrates. The activity of the hydrolase is limited to the conversion of the nitrile group to the carboxamide group. Hydrolase activity is observed in cell-frre estracts of S. rimosus before toyocamycin production begins. The in vivo and in vitro studies demonstrate that toyocamycin is not a precursor for tubercidin. The experimental evidence strongly suggests that there must be a branch point in the biosynthesis of the pyrrolopyrimidine nucleoside antibiotics.