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.
Yan
L
L
Department of Protein Engineering, Institute of Biophysics, Chinese Academy of Sciences, Chaoyang District, Beijing, People's Republic of China.
Wu
S
S
Li
H G
HG
Li
J H
JH
Wong
R N
RN
Shi
Q L
QL
Dong
Y C
YC
D000225:Adenine / Q000032:analysis / Q000737:chemistry; D000249:Adenosine Monophosphate / Q000737:chemistry; D019380:Anti-HIV Agents / Q000737:chemistry*; D000972:Antineoplastic Agents, Phytogenic / Q000737:chemistry*; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular; D003460:Crystallization; D020459:Genes, rRNA / Q000502:physiology*; D006026:Glycoside Hydrolases / Q000378:metabolism*; D015394:Molecular Structure; D017354:Point Mutation; D020033:Protein Isoforms; D011994:Recombinant Proteins; D015978:Trichosanthin / Q000737:chemistry*; D014443:Tyrosine
D019380:Anti-HIV Agents; D000972:Antineoplastic Agents, Phytogenic; D020033:Protein Isoforms; D011994:Recombinant Proteins; D000249:Adenosine Monophosphate; D014443:Tyrosine; D015978:Trichosanthin; D006026:Glycoside Hydrolases; D000225:Adenine