Abstract
Type II DNA topoisomerases are essential enzymes that catalyze topological rearrangement of double-stranded DNA using the free energy generated by ATP hydrolysis. Bacterial DNA gyrase is a prototype of this family and is composed of two subunits (GyrA, GyrB) that form a GyrA2GyrB2 heterotetramer. The N-terminal 43-kDa fragment of GyrB (GyrB43) from E. coli comprising the ATPase and the transducer domains has been studied extensively. The dimeric fragment is competent for ATP hydrolysis and its structure in complex with the substrate analog AMPPNP is known. Here, we have determined the remaining conformational states of the enzyme along the ATP hydrolysis reaction path by solving crystal structures of GyrB43 in complex with ADP⋅BeF3, ADP⋅Pi, and ADP. Upon hydrolysis, the enzyme undergoes an obligatory 12° domain rearrangement to accommodate the 1.5 Å increase in distance between the γ- and β-phosphate of the nucleotide within the sealed binding site at the domain interface. Conserved residues from the QTK loop of the transducer domain (also part of the domain interface) couple the small structural change within the binding site with the rigid body motion. The domain reorientation is reflected in a significant 7 Å increase in the separation of the two transducer domains of the dimer that would embrace one of the DNA segments in full-length gyrase. The observed conformational change is likely to be relevant for the allosteric coordination of ATP hydrolysis with DNA binding, cleavage/re-ligation and/or strand passage.
Highlights
Type II DNA topoisomerases are essential enzymes that catalyze topological rearrangement of double-stranded DNA to maintain chromosomes in an appropriate state
DNA gyrase introduces negative supercoils into covalently closed double-stranded DNA (dsDNA) molecules using the free energy generated by ATP hydrolysis
The fragment was found to be competent for ATP hydrolysis as assessed by an FPLC based nucleotide quantification method [24] yielding kcat and Km values of 2.7?1023 s21 and 0.45 mM, respectively
Summary
Type II DNA topoisomerases are essential enzymes that catalyze topological rearrangement of double-stranded DNA (dsDNA) to maintain chromosomes in an appropriate state. DNA gyrase introduces negative supercoils into covalently closed dsDNA molecules using the free energy generated by ATP hydrolysis. The current view on the enzymatic mechanism is the following two-gate mechanism (reviewed in [1,2,3,4,5]). The enzymatic cycle starts by the binding of a segment of double-stranded DNA (gate-segment, G) to the DNA-gate, followed by the trapping of another segment (transfer-segment, T) through the ATP-actuated closure, i.e. dimerization, of the N-gate. Subsequent cleavage of the Gsegment and opening of the DNA-gate allows transfer of the T-
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