Abstract
The alternative oxidase (AOX) is widespread in plants, fungi, and some protozoa. While the general structure of the AOX remains consistent, its overall activity, sources of kinetic activation and their sensitivity to inhibitors varies between species. In this study, the recombinant Trypanosoma brucei AOX (rTAO) and Arabidopsis thaliana AOX1A (rAtAOX1A) were expressed in the Escherichia coli ΔhemA mutant FN102, and the kinetic parameters of purified AOXs were compared. Results showed that rTAO possessed the highest Vmax and Km for quinol-1, while much lower Vmax and Km were observed in the rAtAOX1A. The catalytic efficiency (kcat/Km) of rTAO was higher than that of rAtAOX1A. The rTAO also displayed a higher oxygen affinity compared to rAtAOX1A. It should be noted that rAtAOX1a was sensitive to α-keto acids while rTAO was not. Nevertheless, only pyruvate and glyoxylate can fully activate Arabidopsis AOX. In addition, rTAO and rAtAOX1A showed different sensitivity to AOX inhibitors, with ascofuranone (AF) being the best inhibitor against rTAO, while colletochlorin B (CB) appeared to be the most effective inhibitor against rAtAOX1A. Octylgallate (OG) and salicylhydroxamic acid (SHAM) are less effective than the other inhibitors against protist and plant AOX. A Caver analysis indicated that the rTAO and rAtAOX1A differ with respect to the mixture of polar residues lining the hydrophobic cavity, which may account for the observed difference in kinetic and inhibitor sensitivities. The data obtained in this study are not only beneficial for our understanding of the variation in the kinetics of AOX within protozoa and plants but also contribute to the guidance for the future development of phytopathogenic fungicides.
Highlights
The alternative oxidase (AOX) is a terminal oxidase that introduces a branch point in the mitochondrial electron transport chain (ETC) at the ubiquinone pool (Moore and Siedow, 1991)
The mature AOX sequences were removed on a NdeI-BamHI fragment and ligated to NdeI-BamHI digested pET15b, in which the 6×His tag was replaced by a Twin-Strep tag to produce the expression construct pET.recombinant Trypanosoma brucei AOX (rTAO)/rAtAOX1A
RTAO and rAtAOX1A lacking the mitochondrial targeting signal sequence and fused to a twin-strep tag were expressed in the E. coli FN102 heme-deficient strain
Summary
The alternative oxidase (AOX) is a terminal oxidase that introduces a branch point in the mitochondrial electron transport chain (ETC) at the ubiquinone pool (Moore and Siedow, 1991). The AOX catalyzes the oxidation of ubiquinol while reducing O2 to H2O, but unlike cytochrome c oxidase (complex IV), it does not translocate protons from the matrix to the intermembrane space (Vanlerberghe and McIntosh, 1997; Moore and Albury, 2008; Moore et al, 2013). It is assumed that the AOX occurs in prokaryotes and enters the eukaryotic cell lineage through major symbiotic events, but AOX activity is quite different in prokaryotes and eukaryotes and is regulated by different effectors (McDonald, 2008; Moore and Albury, 2008)
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