Role of TYR70 in the N-glycosidase activity of neo-trichosanthin.

Trichosanthin (TCS) is a type I ribosome-inactivating protein (RIP) which possesses rRNA N-glycosidase activity. TCS has long been used as an abortifacient in China. In recent years, its immunomodulatory, anti-tumor and anti-HIV properties have attracted more and more attention. An isoform of trichosanthin, neo-trichosanthin (n-TCS), has been cloned and expressed as recombinant protein. The biochemical studies revealed that n-TCS has virtually the same rRNA N-glycosidase activity as TCS. The crystal structure of n-TCS is similar to TCS. The crystal of Y70A n-TCS, the mutant of recombinant n-TCS, was soaked in sodium citrate buffer (pH 5.5) containing 25% KCl and AMP (10 mg/ml) prior to data collection. After structure determination and refinement, no electron density corresponding to adenine can be detected around the active pocket. Furthermore, the reaction products of Y70A n-TCS and AMP incubated at various reaction times were analyzed using HPLC. No adenine can be detected. These results suggest that Tyr70 is crucial to n-TCS for its substrate recognition, binding and perhaps N-glycosidase activity.