Mutational analysis of Escherichia coli topoisomerase IV. Identification of a region of ParE involved in covalent catalysis

The products of three dominant-negative alleles of parE, encoding the ATP-b inding subunit of topoisomerase TV (Topo TV), were purified and their activ ities characterized when reconstituted with ParC to form Topo IV. The abili ty of the ParE E418K, ParE G419D, and ParE G442D mutant Topo IVs to bind DN A, hydrolyze ATP, and close their ATP-dependent clamp was relatively unaffe cted. However, their ability to relax negatively supercoiled DNA was compro mised significantly. This could be attributed to severe defects in covalent complex formation between ParC and DNA. Thus, these residues, which are fa r from the active site Tyr of ParC, contribute to covalent catalysis, This indicates that a dramatic conformational rearrangement of the protein likel y occurs subsequent to the binding of the G: segment at the DNA gate and pr ior to its opening.