AUTHOR=Xu Fei , Copsey Alice C. , Young Luke , Barsottini Mario R. O. , Albury Mary S. , Moore Anthony L. 

TITLE=Comparison of the Kinetic Parameters of Alternative Oxidases From Trypanosoma brucei and Arabidopsis thaliana—A Tale of Two Cavities

JOURNAL=Frontiers in Plant Science

VOLUME=Volume 12 - 2021

YEAR=2021

URL=https://www.frontiersin.org/journals/plant-science/articles/10.3389/fpls.2021.744218

DOI=10.3389/fpls.2021.744218

ISSN=1664-462X

ABSTRACT=<p>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 <italic>Trypanosoma brucei</italic> AOX (rTAO) and <italic>Arabidopsis thaliana</italic> AOX1A (rAtAOX1A) were expressed in the <italic>Escherichia coli</italic> Δ<italic>hemA</italic> mutant FN102, and the kinetic parameters of purified AOXs were compared. Results showed that rTAO possessed the highest <italic>V</italic><sub>max</sub> and <italic>K</italic><sub>m</sub> for quinol-1, while much lower <italic>V</italic><sub>max</sub> and <italic>K</italic><sub>m</sub> were observed in the rAtAOX1A. The catalytic efficiency (<italic>k</italic><sub>cat</sub>/<italic>K</italic><sub>m</sub>) 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.</p>