Functional expression of the ascofuranone-sensitive Trypanosoma brucei brucei alternative oxidase in the cytoplasmic membrane of Escherichia coli

Trypanosome alternative oxidase (TAO) is the terminal oxidase of the respir atory chain of long slender bloodstream forms (LS forms) of African trypano soma, which causes sleeping sickness in human and nagana in cattle. TAO is a cytochrome-independent; cyanide-insensitive quinol oxidase and these prop erties are quite different from those of the bacterial quinol oxidase which belongs to the heme-copper terminal oxidase superfamily. Only little infor mation concerning the molecular structure and enzymatic features of TAO hav e been available, whereas the bacterial enzyme has been well characterized. In this study, a cDNA encoding TAO from Trypanosoma brucei brucei was clon ed into the expression vector pET15b (pTAO) and recombinant TAO was express ed in Eschericizia coli. The growth of the transformant carrying pTAO was c yanide-resistant. A peptide with a molecular mass of 37 kDa was found in th e cytoplasmic membrane of E. coli, and was recognized by antibodies against plant-type alternative oxidases from Sauriomalum guttatum and Hansenula an omala. Both the ubiquinol oxidase and succinate oxidase activities found in the membrane of the transformant were insensitive to cyanide, while those of the control strain, which contained Vector alone, were inhibited. This c yanide-insensitive growth of the E. coli carrying pTAO was inhibited by the addition of ascofuranone, a potent and specific inhibitor of TAO ubiquinol oxidase. The ubiquinol oxidase activity of the membrane from the transform ant was sensitive to ascofuranone. These results clearly show the functiona l expression of TAO in E. coli and indicate that ubiquinot-8 in the E. coil membrane is able to serve as an electron donor to the recombinant enzyme a nd confer cyanide-resistant and ascofuranone-sensitive growth to E. coil. T his system will facilitate the biochemical characterization of the novel te rminal oxidase, TAO, and the understanding on the mechanism of the trypanoc idal effect of ascofuranone. (C) 1999 Elsevier Science Inc. All rights rese rved.