tailieunhanh - Báo cáo khoa học: Unique features of recombinant heme oxygenase of Drosophila melanogaster compared with those of other heme oxygenases studied

We cloned a cDNA for aDrosophila melanogasterhomo-logue ofmammalian heme oxygenase (HO) and constructed a bacterial expression system of a truncated, soluble form ofD. melanogasterHO (DmDHO). The purified DmDHO degraded hemin to biliverdin, CO and iron in the presence of reducing systems such as NADPH/cytochrome P450 reductase and sodium ascorbate, although the reaction rate was slower than that of mammalian HOs. Some properties of DmHO, however, are quite different from other known HOs. | Eur. J. Biochem. 271 1713-1724 2004 FEBS 2004 doi Unique features of recombinant heme oxygenase of Drosophila melanogaster compared with those of other heme oxygenases studied Xuhong Zhang1 Michihiko Sato2 Masanao Sasahara1 Catharina T. Migita3 and Tadashi Yoshida1 1 Department of Biochemistry and 2Central Laboratory for Research and Education Yamagata University School of Medicine Japan 3Department of Biological Chemistry Faculty of Agriculture Yamaguchi University Japan We cloned a cDNA for a Drosophila melanogaster homologue of mammalian heme oxygenase HO and constructed a bacterial expression system of a truncated soluble form of D. melanogaster HO DmAHO . The purified DmAHO degraded hemin to biliverdin CO and iron in the presence of reducing systems such as NADPH cytochrome P450 reductase and sodium ascorbate although the reaction rate was slower than that of mammalian HOs. Some properties of DmHO however are quite different from other known HOs. Thus DmAHO bound hemin stoichiometrically to form a hemin-enzyme complex like other HOs but this complex did not show an absorption spectrum of hexacoordinated heme protein. The absorption spectrum of the ferric complex was not influenced by changing the pH of the solution. Interestingly an EPR study revealed that the iron of heme was not involved in binding heme to the enzyme. Hydrogen peroxide failed to convert it into verdoheme. A spectrum of the ferrous-CO form of verdoheme was not detected during the reaction from hemin under oxygen and CO. Degradation of hemin catalyzed by DmAHO yielded three isomers of biliverdin of which biliverdin II a and two other isomers IXb and IXỖ accounted for 75 and 25 respectively. Taken together we conclude that although DmHO acts as a real HO in D. melanogaster its active-site structure is quite different from those of other known HOs. Keywords biliverdin Drosophila melanogaster heme oxygenase insect NADPH cytochrome P450 reductase. Heme oxygenase HO