Effects of mutating specific residues present near the amino terminus of 2'-5'-oligoadenylate synthetase.
In this study, we investigated the role of specific amino acid residues present near the amino terminus of the 9-2 isozyme of 2'-5'-oligoadenylate synthetase. In vitro expression of deletion mutants showed that residues 1-9 are required for enzyme activity. Within this region, residues 3, 7, and 8 were found to be conserved among all known isozymes of 2'-5'-oligoadenylate synthetase. Mutation of these residues singly or in combination resulted in partial or total loss of enzyme activity. Substitution of the proline residue at position 7 by different residues caused a partial or complete loss of activity. The properties of the inactive P7Q mutant were further explored by expressing the protein in bacteria. The bacterially expressed protein was also enzymatically inactive. The mutant protein could bind the substrate ATP and the activator double-stranded RNA normally. Oligomerization properties of the protein were examined by an affinity-based interaction assay and by glycerol gradient centrifugation; there was no detectable difference between the wild type and the P7Q mutant. These results demonstrated the importance of the proline residue at position 7 in conferring enzyme activity to the protein without affecting its other properties.
Ghosh
A
A
Department of Molecular Biology, Research Institute, The Cleveland Clinic Foundation, Cleveland, Ohio 44195, USA.
Desai
S Y
SY
Sarkar
S N
SN
Ramaraj
P
P
Ghosh
S K
SK
Bandyopadhyay
S
S
Sen
G C
GC
D015088:2',5'-Oligoadenylate Synthetase / Q000235:genetics / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism; D001704:Biopolymers; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D012330:RNA, Double-Stranded / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D017384:Sequence Deletion
D001704:Biopolymers; D012330:RNA, Double-Stranded; D016601:RNA-Binding Proteins; D000255:Adenosine Triphosphate; D015088:2',5'-Oligoadenylate Synthetase