title: Crisscross enzymatic reaction between the two molecules in the active dimeric P69 form of the 2'-5' oligodenylate synthetase.
abstract: 2'-5' oligoadenylate (2-5 (A)) synthetases are major components of the antiviral pathways induced by interferons. In the presence of double-stranded RNA, they polymerize ATP to form 2-5 (A) oligomers that, in turn, activate the latent ribonuclease RNase L, causing mRNA degradation. These enzymes, unlike other nucleotidyl transferases, catalyze 2'-5', not 3'-5', phosphodiester bond formation between substrates bound to the acceptor and donor sites. Moreover, unlike other members of this extended family, the P69 isozyme of 2-5 (A) synthetase functions as a homodimer. Here, we report that the need for P69 dimerization is because of a crisscross enzyme reaction joining two substrate molecules bound to two opposite subunits. Consequently, although homodimers of mutants in the previously identified acceptor site, the donor site, or the catalytic site were inactive, selective heterodimers of the mutants were active because of subunit complementation. The catalytic site had to be present in the same subunit that contained the acceptor site, whereas the donor site had to be provided by the other subunit. These results allowed us to design a mutant protein that acted as a dominant-negative inhibitor of wt P69 but not of another isozyme of 2-5 (A) synthetase.
authors.affiliation: Department of Molecular Biology, The Lerner Research Institute, Cleveland Clinic Foundation, Cleveland, Ohio 44195, USA.
authors.forename: Saumendra N
authors.initials: SN
authors.lastname: Sarkar
authors.forename: Srabani
authors.initials: S
authors.lastname: Pal
authors.forename: Ganes C
authors.initials: GC
authors.lastname: Sen
mesh_terms: D015088:2',5'-Oligoadenylate Synthetase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites; D019281:Dimerization; D005816:Genetic Complementation Test; D006801:Humans; D007527:Isoenzymes / Q000235:genetics / Q000378:metabolism*; D015394:Molecular Structure; D016297:Mutagenesis, Site-Directed; D009690:Nucleic Acid Conformation; D021122:Protein Subunits / Q000235:genetics / Q000378:metabolism; D012330:RNA, Double-Stranded / Q000378:metabolism
chemical_list: D007527:Isoenzymes; D021122:Protein Subunits; D012330:RNA, Double-Stranded; D015088:2',5'-Oligoadenylate Synthetase