Bacteria frequently possess two type IIA DNA topoisomerases, gyrase and topo IV, which maintain chromosome topology by variously supercoiling, relaxing, and disentangling DNA. DNA recognition and functional output is thought to be controlled by the C-terminal domain (CTD) of the topoisomerase DNA binding subunit (GyrA/ParC). The deeply rooted organism
Aquifex aeolicus
encodes one type IIA topoisomerase conflictingly categorized as either DNA gyrase or topo IV. To resolve this enzyme’s catalytic properties and heritage, we conducted a series of structural and biochemical studies on the isolated GyrA/ParC CTD and the holoenzyme. Whereas the CTD displays a global structure similar to that seen in bone fide GyrA and ParC paralogs, it lacks a key functional motif (the “GyrA-box”) and fails to wrap DNA. Biochemical assays show that the
A. aeolicus
topoisomerase cannot supercoil DNA, but robustly removes supercoils and decatenates DNA, two hallmark activities of topo IV. Despite these properties, phylogenetic analyses place all functional domains except the CTD squarely within a gyrase lineage, and the
A. aeolicus
GyrB subunit is capable of supporting supercoiling with
Escherichia coli
GyrA, but not DNA relaxation with
E. coli
ParC. Moreover, swapping the
A. aeolicus
GyrA/ParC CTD with the GyrA CTD from
Thermotoga maritima
creates an enzyme that negatively supercoils DNA. These findings identify
A. aeolicus
as the first bacterial species yet found to exist without a functional gyrase, and suggest an evolutionary path for generation of bacterial type IIA paralogs.