REVERSE GYRASE FROM METHANOPYRUS-KANDLERI - RECONSTITUTION OF AN ACTIVE EXTREMOZYME FROM ITS 2 RECOMBINANT SUBUNITS

Reverse gyrases are ATP-dependent type I 5'-topoisomerases that positi vely supercoil DNA. Reverse gyrase from Methanopyrus kandleri is uniqu e as the first heterodimeric type I 5'-topoisomerase described, con si sting of a 138-kDa subunit involved in the hydrolysis of ATP (RgyB) an d a 43-kDa subunit that forms the covalent complex with DNA during the topoisomerase reaction (RgyA). Here we report the reconstitution of a ctive reverse gyrase from the two recombinant proteins overexpressed i n Escherichia coli. Both proteins have been purified by column chromat ography 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 R gyA subunits form a stable heterodimer only after being heated togethe r. This is the first case in which it has been possible to reconstitut e an active heterodimeric enzyme of a hyperthermophilic prokaryote fro m recombinant proteins.