Reverse gyrase from Methanopyrus kandleri. Reconstitution of an active extremozyme from its two recombinant subunits.
Reverse gyrases are ATP-dependent type I 5'-topoisomerases that positively supercoil DNA. Reverse gyrase from Methanopyrus kandleri is unique as the first heterodimeric type I 5'-topoisomerase described, consisting of a 138-kDa subunit involved in the hydrolysis of ATP (RgyB) and a 43-kDa subunit that forms the covalent complex with DNA during the topoisomerase reaction (RgyA). Here we report the reconstitution of active reverse gyrase from the two recombinant proteins overexpressed in Escherichia coli. Both proteins have been purified by column chromatography to >90% homogeneity. RgyB has a DNA-dependent ATPase activity at high temperature (80 degrees C) and is independent of the presence of RgyA. RgyA alone has no detectable activity. The addition of RgyA to RgyB reconstitutes positive supercoiling activity, but the RgyB and RgyA subunits form a stable heterodimer only after being heated together. This is the first case in which it has been possible to reconstitute an active heterodimeric enzyme of a hyperthermophilic prokaryote from recombinant proteins.
Krah
R
R
Laboratory of Molecular Biology, NIDDK, National Institutes of Health, Bethesda, Maryland 20892-0540, USA.
O'Dea
M H
MH
Gellert
M
M
D000251:Adenosine Triphosphatases / Q000235:genetics / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D004265:DNA Helicases / Q000235:genetics / Q000378:metabolism; D004264:DNA Topoisomerases, Type I; D004250:DNA Topoisomerases, Type II / Q000737:chemistry* / Q000378:metabolism; D019281:Dimerization; D004926:Escherichia coli; D019605:Euryarchaeota / Q000201:enzymology*; D008274:Magnesium / Q000378:metabolism; D008970:Molecular Weight; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism
D011994:Recombinant Proteins; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases; D004265:DNA Helicases; C041813:DNA reverse gyrase; D004264:DNA Topoisomerases, Type I; D004250:DNA Topoisomerases, Type II; D008274:Magnesium