DNA gyrase is a bacterial type II topoisomerase which couples the free energy of ATP hydrolysis to the introduction of negative supercoils into DNA. Amino acids in proximity to bound nonhydrolyzable ATP analog (AMP ⅐ PNP) or novobiocin in the gyrase B (GyrB) subunit crystal structures were examined for their roles in enzyme function and novobiocin resistance by site-directed mutagenesis. Purified Escherichia coli GyrB mutant proteins were complexed with the gyrase A subunit to form the functional A 2 B 2 gyrase enzyme. Mutant proteins with alanine substitutions at residues E42, N46, E50, D73, R76, G77, and I78 had reduced or no detectable ATPase activity, indicating a role for these residues in ATP hydrolysis. Interestingly, GyrB proteins with P79A and K103A substitutions retained significant levels of ATPase activity yet demonstrated no DNA supercoiling activity, even with 40-fold more enzyme than the wild-type enzyme, suggesting that these amino acid side chains have a role in the coupling of the two activities. All enzymes relaxed supercoiled DNA to the same extent as the wild-type enzyme did, implying that only ATP-dependent reactions were affected. Mutant genes were examined in vivo for their abilities to complement a temperature-sensitive E. coli gyrB mutant, and the activities correlated well with the in vitro activities. We show that the known R136 novobiocin resistance mutations bestow a significant loss of inhibitor potency in the ATPase assay. Four new residues (D73, G77, I78, and T165) that, when changed to the appropriate amino acid, result in both significant levels of novobiocin resistance and maintain in vivo function were identified in E. coli.Bacterial DNA gyrase is an established target for the development of new antibiotics (19, 28), being an essential type II topoisomerase involved in maintenance of the negative superhelicity of the chromosome during replication and transcription. The enzyme consists of two subunits (subunits A and B) that combine into the heterotetrameric A 2 B 2 complex to form the functional enzyme. The GyrB subunit is comprised of an N-terminal domain (43 kDa) which contains the ATPase active site and a C-terminal domain (47 kDa) which is involved in the interaction with both the GyrA subunit and DNA. A DNA supercoiling model for gyrase was proposed on the basis of the work by Mizuuchi et al. (31) and Roca and Wang (37,38). Many aspects of the gyrase supercoiling model have been examined experimentally (22-24, 35, 46, 48-50). In brief, gyrase binds to a short segment of DNA and cleaves the doublestranded DNA, which creates a DNA gate. These activities are mediated via GyrA subunits. DNA contiguous with the DNA gate is wrapped around the GyrA subunits and presents a segment of DNA (termed the transport segment or T segment) to the open N-terminal GyrB subunits (termed the ATP-operated clamp). Upon ATP binding, the ATP-operated clamp closes, capturing the T segment. The DNA gate opens, pulling the broken ends of the DNA gate apart and facilitating the passage of the T seg...