title: Crystal structure of ribonuclease Ms (as a ribonuclease T1 homologue) complexed with a guanylyl-3',5'-cytidine analogue.
abstract: A ribonuclease T1 homologue, ribonuclease Ms (RNase Ms) from Aspergillus saitoi, has been crystallized as a complex with a substrate analogue GfpC where the 2'-hydroxyl (2'-OH) group of guanosine in guanylyl-3',5'-cytidine (GpC) is replaced by the 2'-fluorine (2'-F) atom to prevent transesterification. The crystal structure of the complex was solved at 1.8-A resolution to a final R-factor of 0.204. The role of His92 (RNase T1 numbering) as the general acid catalyst was confirmed. Of the two alternative candidates for a general base to abstract a proton from the 2'-OH group, His40 and Glu58 were found close to the 2'-F atom, making the decision between the two groups difficult. We then superposed the active site of the RNase Ms/GfpC complex with that of pancreatic ribonuclease S (RNase S) complexed with a substrate analogue UpcA, a phosphonate analogue of uridylyl-3',5'-adenosine (UpA), and found that His12 and His119 of RNase A almost exactly coincided with Glu58 and His92, respectively, of RNase Ms. Similar superposition with a prokaryotic microbial ribonuclease, RNase St [Nakamura, K. T., Iwahashi, K., Yamamoto, Y., Iitaka, Y., Yoshida, N., & Mitsui, Y. (1982) Nature 299, 564-566], also indicated Glu58 as a general base. Thus the present comparative geometrical studies consistently favor, albeit indirectly, the traditional as well as the most recent notion [Steyaert, J., Hallenga, K., Wyns, L., & Stanssens, P. (1990) Biochemistry 29, 9064-9072] that Glu58, rather than His40, must be the general base catalyst in the intact enzymes of the RNase T1 family.
authors.affiliation: Department of BioEngineering, Nagaoka University of Technology, Japan.
authors.forename: T
authors.initials: T
authors.lastname: Nonaka
authors.forename: K T
authors.initials: KT
authors.lastname: Nakamura
authors.forename: S
authors.initials: S
authors.lastname: Uesugi
authors.forename: M
authors.initials: M
authors.lastname: Ikehara
authors.forename: M
authors.initials: M
authors.lastname: Irie
authors.forename: Y
authors.initials: Y
authors.lastname: Mitsui
mesh_terms: D000595:Amino Acid Sequence; D001230:Aspergillus / Q000201:enzymology; D003460:Crystallization; D015226:Dinucleoside Phosphates / Q000378:metabolism*; D004722:Endoribonucleases / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D005583:Fourier Analysis; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011485:Protein Binding; D011487:Protein Conformation; D012260:Ribonucleases / Q000737:chemistry / Q000378:metabolism; D013329:Structure-Activity Relationship; D014961:X-Ray Diffraction / Q000379:methods
chemical_list: D015226:Dinucleoside Phosphates; C074435:2'-deoxy-2'-fluoroguanylyl-3',5'-cytidine; D004722:Endoribonucleases; D012260:Ribonucleases; C022543:ribonuclease T(2)