Complex of ribonuclease Sa with a cyclic nucleotide and a proposed model for the reaction intermediate

Abstract

The structure of the complex of ribonuclease from Streptomyces aureofaciens (RNase Sa) with ex0 guanosine 2',3'-cyclophosphorothioate has been refined against 0.2-nm resolution synchrotron data using, as a starting model, coordinates from the RNase Sa: 2'-GMP complex. The refinement was based on all data over 1.0-0.2 nm and converged to a crystallographic R factor of 11.9%. This is the first structure of a microbial ribonuclease complexed with a 2',3'-cyclophosphorothioate, which is a thio analogue of the intermediate of the two-step reaction. However, ex0 guanosine 2',3'-cyclophosphorothioate is bound in a non-functional mode and is not hydrolysed. This structure therefore does not provide direct evidence on the identity of the amino acid residues responsible for catalytic cleavage of the substrate. However, based on present and previous results, a plausible model is proposed for the complex of the cyclic intermediate which acts as substrate for the second step of the catalysis.

Three Dimensional Structure of the MqsR:MqsA Complex: A Novel TA Pair Comprised of a Toxin Homologous to RelE and an Antitoxin with Unique Properties

“…RNase Sa (orange; PDBID 1RSN [48]), a canonical bacterial RNase, is also shown. The bacterial RNase fold is characterized by a central, twisted β-sheet and adjacent α–helix and is conserved among all four proteins.…”

“…8A). In microbial RNases, such as RNase Sa, RNA binding is mediated by polar (Q38) and aromatic (Y86) residues while RNA cleavage is catalyzed by a histidine (H85) and glutamic acid (E54) residue [47],[48]. While the catalytic histidine and glutamic acid are conserved in YoeB (H83, E46), which has intrinsic endoribonuclease activity [33], they are not found in RelE, which functions as a ribosome-dependent RNase [23],[45].…”

Ribonucleases as potential modalities in anticancer therapy

“…RNase Sa is even smaller protein (96 residues) (Shlyapnikov et al, 1986). Its crystallographic structure, also in complexes with ligands was determined (Sevcik et al, 1991, 1993). The structure of its isoenzyme Sa3 was also refined (Sevcik et al, 2002).…”

Crystal structure reveals two alternative conformations in the active site of ribonuclease Sa2

“…The aromatic ring of Tyr87C is positioned in a plane very close to that of the mononucleotide base and interacts with Tyr87A and Phe39A, which form the bottom of the active site. In addition, Tyr87C OH forms a hydrogen bond with the amide NH group of Arg42A similar to that formed by the O6 atom of mononucleotide bases in the complexes with RNase Sa (Sevcik et al, 1991;Sevcik, Hill et al, 1993;Sevcik, Zegers et al, 1993). Molecules A and C are bound to one another in the crystal by 18 hydrogen bonds, four of them mediated by water molecules, and by the burial of Tyr87C.…”

“…The structures of the enzyme and its complexes with guanosine-3 H -monophosphate (3 H -GMP; Sevcik et al, 1991), guanosine-2 H -monophosphate (2 H -GMP; Sevcik, Hill et al, 1993) and guanosine-2 H ,3 H -cyclophosphorothioate (Sevcik, Zegers et al, 1993) have been determined at high resolution. The structure of the free enzyme has been re®ned at 1.2 A Ê resolution and subsequently at 1.0 A Ê resolution (S AE evc Ïõ Âk, Lamzin et al, 2002).…”