Active-site residues of Escherichia coli DNA gyrase required in coupling ATP hydrolysis to DNA supercoiling and amino acid substitutions leading to novobiocin resistance

Abstract

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. 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.

FIG. 1.

Locations of selected amino acids in the E. coli GyrB ATP binding site relative to AMP · PNP and novobiocin. (A) AMP · PNP complexed in the E. coli N-terminal 43-kDa fragment based on the structural data for the sequence in the Protein Data Bank with identification number 1EI1. (B) Novobiocin complexed in the E. coli N-terminal 24-kDa fragment (novobiocin-resistant mutant R136H) based on the structural data for the sequence in the Protein Data Bank with identification number 1AJ6. The locations of the side chains for residues E42, N46, E50, D73, R76, G77, I78, P79, K103, V120, R136 or H136, T165, and V167 are shown and labeled in both panels A and B. The α-carbon of the mutant residues is shown in gold. The protein backbone is shown as a thin green line.

FIG. 1.

Locations of selected amino acids in the E. coli GyrB ATP binding site relative to AMP · PNP and novobiocin. (A) AMP · PNP complexed in the E. coli N-terminal 43-kDa fragment based on the structural data for the sequence in the Protein Data Bank with identification number 1EI1. (B) Novobiocin complexed in the E. coli N-terminal 24-kDa fragment (novobiocin-resistant mutant R136H) based on the structural data for the sequence in the Protein Data Bank with identification number 1AJ6. The locations of the side chains for residues E42, N46, E50, D73, R76, G77, I78, P79, K103, V120, R136 or H136, T165, and V167 are shown and labeled in both panels A and B. The α-carbon of the mutant residues is shown in gold. The protein backbone is shown as a thin green line.