Crystal Structures of Escherichia coli Topoisomerase IV ParE Subunit (24 and 43 Kilodaltons): a Single Residue Dictates Differences in Novobiocin Potency against Topoisomerase IV and DNA Gyrase

Abstract

Topoisomerase IV and DNA gyrase are related bacterial type II topoisomerases that utilize the free energy from ATP hydrolysis to catalyze topological changes in the bacterial genome. The essential function of DNA gyrase is the introduction of negative DNA supercoils into the genome, whereas the essential function of topoisomerase IV is to decatenate daughter chromosomes following replication. Here, we report the crystal structures of a 43-kDa N-terminal fragment of Escherichia coli topoisomerase IV ParE subunit complexed with adenylyl-imidodiphosphate at 2.0-Å resolution and a 24-kDa N-terminal fragment of the ParE subunit complexed with novobiocin at 2.1-Å resolution. The solved ParE structures are strikingly similar to the known gyrase B (GyrB) subunit structures. We also identified single-position equivalent amino acid residues in ParE (M74) and in GyrB (I78) that, when exchanged, increased the potency of novobiocin against topoisomerase IV by nearly 20-fold (to 12 nM). The corresponding exchange in gyrase (I78 M) yielded a 20-fold decrease in the potency of novobiocin (to 1.0 M). These data offer an explanation for the observation that novobiocin is significantly less potent against topoisomerase IV than against DNA gyrase. Additionally, the enzyme kinetic parameters were affected. In gyrase, the ATP K m increased Ϸ5-fold and the V max decreased Ϸ30%. In contrast, the topoisomerase IV ATP K m decreased by a factor of 6, and the V max increased Ϸ2-fold from the wild-type values. These data demonstrate that the ParE M74 and GyrB I78 side chains impart opposite effects on the enzyme's substrate affinity and catalytic efficiency.Type II topoisomerases catalyze the interconversion of DNA topoisomers by transporting one DNA segment through another. Bacterial genomes encode two type II topoisomerases, DNA gyrase and topoisomerase IV (TopoIV), that function in DNA replication. DNA gyrase is unique in coupling the free energy of ATP hydrolysis to the introduction of negative supercoils into DNA. In the absence of the ATP substrate, DNA gyrase can relax negatively supercoiled plasmid DNA. These activities result from the enzyme's ability to wrap (Ϸ150 bp) DNA (23, 31) around itself upon binding the DNA substrate. This DNA wrapping preferentially presents the T-segment (transported DNA segment) to the gyrase-DNA complex so that the introduction of negative supercoils is the primary outcome. In contrast, TopoIV and other eukaryotic type II topoisomerases only bind a Ϸ30-bp region of DNA (20,35). TopoIV utilizes the energy of ATP hydrolysis to decatenate newly replicated chromosomal DNA but also has the ability to relax positive and negative DNA supercoils in an ATP-dependent manner (8,43).In prokaryotes, these type II topoisomerases are composed of two subunits. In Escherichia coli, the gyrase subunits are named A and B and the corresponding TopoIV subunits are named C and E. For each enzyme, these subunits combine into a heterotetrameric (gyrase, A 2 B 2 ; and TopoIV, C 2 E 2 ) complex to form the functional enzymes. I...

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