Solution structure of the catalytic domain of gamma delta resolvase. Implications for the mechanism of catalysis

The site-specific DNA recombinase, gamma delta resolvase, from Escherichia coli catalyzes recombination of res site-containing plasmid DNA to two cate nated circular DNA products. The catalytic domain (residues 1-105), lacking a C-terminal dimerization interface, has been constructed and the NMR solu tion structure of the monomer determined. The RMSD of the NMR conformers fo r residues 2-92 excluding residues 37-45 and 64-73 is 0.41 Angstrom for bac kbone atoms and 0.88 Angstrom for all heavy atoms. The NMR solution structu re of the monomeric catalytic domain (residues 1-105) was found to be forme d by a four-stranded parallel P-sheet surrounded by three helices. The cata lytic domain (residues 1-105), deficient in the C-terminal dimerization dom ain, was monomeric at high salt concentration, but displayed unexpected dim erization at lower ionic strength. The unique solution dimerization interfa ce at low ionic strength was mapped by NMR. With respect to previous crysta l structures of the dimeric catalytic domain (residues 1-140), differences in the average conformation of active-site residues were found at loop 1 co ntaining the catalytic S10 nucleophile, the beta1 strand containing R8, and at loop 3 containing D67, R68 and R71, which are required for catalysis. T he active-site loops display high-frequency and conformational backbone dyn amics and are less well defined than the secondary structures. In the solut ion structure, the D67 side-chain is proximal to the S10 side-chain making the D67 carboxylate group a candidate for activation of S10 through general base catalysis. Four conserved Arg residues can function in the activation of the phosphodiester for nucleophilic attack by the S10 hydroxyl group. A mechanism for covalent catalysis by this class of recombinases is proposed that may be related to dimer interface dissociation. (C) 2001 Academic Pre ss.