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Antimicrobial Agents and Chemotherapy, May 2004, p. 1856-1864, Vol. 48, No. 5
0066-4804/04/$08.00+0     DOI: 10.1128/AAC.48.5.1856-1864.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

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

Steven Bellon, Jonathan D. Parsons, Yunyi Wei,^* Koto Hayakawa, Lora L. Swenson, Paul S. Charifson, Judith A. Lippke, Robert Aldape, and Christian H. Gross

Vertex Pharmaceuticals Incorporated, Cambridge, Massachusetts 02139

Received 10 September 2003/ Returned for modification 30 November 2003/ Accepted 31 January 2004

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 µM). 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 5-fold and the V_max decreased 30%. In contrast, the topoisomerase IV ATP K_m decreased by a factor of 6, and the V_max increased 2-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.

Present address: Amgen Inc., One Kendall Square, Cambridge, MA 02139.

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