Active-Site Residues of Escherichia coli DNA Gyrase Required in Coupling ATP Hydrolysis to DNA Supercoiling and Amino Acid Substitutions Leading to Novobiocin Resistance

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 A2B2 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 double-stranded 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 segment through the gap. To complete the strand-passage cycle, the DNA gate is religated upon closure, the T segment is expelled via opening of the exit gate (C-terminal GyrA subunits), and the ATP-operated clamp reopens upon ATP hydrolysis. These actions by the enzyme change the superhelical state of the DNA substrate. There are known synthetic (e.g., quinolone) and natural product (e.g., coumarin) inhibitors of bacterial gyrase. These two types of antibiotics act on gyrase through different mechanisms; the quinolones inhibit the strand breakage and rejoining function of the A subunit. The coumarin-containing antibiotics, of which novobiocin and its dimer coumermycin A1 are representative examples, inhibit the ATP-dependent strand-passage function of the B subunit (28). Clinical use of quinolone antibiotics is likely to become restricted due to the emergence of resistant organisms (2, 12, 20). The poor activities of the coumarin-containing antibiotics against gram-negative bacteria, the high frequencies of development of resistance to these antibiotics, and the mammalian cytotoxicities of these antibiotics have limited their use in the clinic (28). However, despite the limitations of the coumarin-containing antibiotics, their potent inhibitory activities against gyrase and their antibacterial activities stress the importance of GyrB as a well-validated antibacterial target that has yet to be fully exploited. In addition, inhibitors of GyrB are likely to be effective against bacteria resistant to GyrA inhibitors, because GyrA subunit mutations are expected to have little effect on GyrB function. The emergence of bacterial resistance to the presently used antibiotics is prompting a new wave of drug discovery efforts. Bacteria can acquire antibiotic resistance by multiple mechanisms including a reduction in cell permeability, active efflux of the antibiotic, degradation or modification of the antibiotic, antibiotic sequestration by protein binding, metabolic bypass of the reaction inhibited by the antibiotic, overproduction of the antibiotic target, and target-based resistance (8, 33). In the case of target-based resistance, the protein target is altered such that the inhibitor no longer effectively binds to it (43). Target-based resistance results from inhibitors that make essential binding contacts with amino acid residues that are nonessential for enzyme catalysis or function. Under these circumstances, spontaneous resistant mutants with alterations in these nonessential target residues can readily arise, rendering the antibiotic ineffective. Similarly, the emergence of target-based resistance to inhibitors (e.g., novobiocin) which do not entirely overlap the substrate binding site or which extend into nonessential subsites within the enzyme is also more likely (43). In an effort to design inhibitors of the GyrB subunit ATP binding site and minimize target-based resistance, we were interested in identifying those amino acid residues which are critical for function. We reasoned that target-based resistance to novel GyrB inhibitors which make the strongest contacts with these essential residues may be far less likely and that such inhibitors may have a greater potential for a broad spectrum of action due to the conservation of these residues among different bacterial species. Using site-directed mutagenesis we found seven amino acid residues in the Escherichia coli GyrB ATP active site that are essential for enzyme function in vitro. These seven residues are also essential for in vivo function, as demonstrated by their inability to complement an E. coli gyrB temperature-sensitive mutant. In addition to the known novobiocin resistance mutation at R136 (9, 13, 36, 39), this study identifies four new amino acid positions in E. coli (D73, G77, I78, and T165) that, when altered, allow significant levels of novobiocin resistance while simultaneously maintaining in vivo function.