Crystal structures of the complexes of trichosanthin with four substrate analogs and catalytic mechanism of RNA N-glycosidase.
Four substrate analogs-nicotinamide adenine dinucleotide, adenylyl (3', 5') guanosine, guanylyl (3',5') adenosine, and adenosine 2', 5'-diphosphate-have been used to prepare the complexes with trichosanthin (TCS), a type I ribosome-inactivating protein that possesses the activity of N-glycosidase. The crystal structures of the complexes have been determined and refined at high resolution. The refined structures show that the N-glycosidic bonds of all the four substrate analogues are hydrolyzed and a common structure is shared by the four complexes, in which only adenine, the product of the enzymatic reaction, is bound in the active center. The structure is compared with those of native trichosanthin and a previously reported trichosanthin-NADPH complex in which the N-glycosidic bond is uncleaved. The structural comparison shows that the conformation of Tyr70 obviously differs from those in the latter two structures, i.e., the side chain of Tyr70 is rotated along its Cbeta-Cgamma bond by approximately 70 degrees. The water molecule found to be preassociated with the N-glycosidic bond in the TCS-NADPH complex structure and proposed to be the water candidate responsible for hydrolyzing the N-glycosidic bond disappears in the trichosanthin-product complex structure. Based on the comparison of the three structures representing the different stages of the enzymatic reaction, the catalytic mechanism of RNA N-glycosidase has been further elucidated. Proteins 2000;39:37-46.
Gu
Y J
YJ
Shanghai Institute of Organic Chemistry, Shanghai, China.
Xia
Z X
ZX
D000244:Adenosine Diphosphate / Q000737:chemistry / Q000378:metabolism; D001665:Binding Sites; D002384:Catalysis; D018360:Crystallography, X-Ray / Q000379:methods; D015226:Dinucleoside Phosphates / Q000737:chemistry / Q000378:metabolism; D006860:Hydrogen Bonding; D008958:Models, Molecular; D015394:Molecular Structure; D009699:N-Glycosyl Hydrolases / Q000737:chemistry* / Q000378:metabolism*; D009243:NAD / Q000737:chemistry / Q000378:metabolism; D017433:Protein Structure, Secondary; D054788:Ribosome Inactivating Proteins; D013379:Substrate Specificity; D015978:Trichosanthin / Q000737:chemistry* / Q000378:metabolism*
D015226:Dinucleoside Phosphates; D009243:NAD; C031924:adenylyl-(3'-5')-guanosine; D015978:Trichosanthin; D000244:Adenosine Diphosphate; C013011:guanine ribonucleotidyl-(3'-5')-adenosine; D009699:N-Glycosyl Hydrolases; D054788:Ribosome Inactivating Proteins