Purification and kinetic characterization of recombinant alternative oxidase from Trypanosoma brucei brucei

Kido, Yasutoshi; Sakamoto, Kimitoshi; Nakamura, Kôsuke; Harada, Masayoshi; Suzuki, Takashi; Yabu, Yoshisada; Saimoto, Hiroyuki; Yamakura, Fumiyuki; Ohmori, Daijiro; Moore, Anthony L.; Harada, Shigeharu; Kita, Kiyoshi

doi:10.1016/j.bbabio.2009.12.021

Cited by 50 publications

(88 citation statements)

References 56 publications

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“…We have recently established protocols to prepare highly purified and stable TAO, which has enabled us to crystallize the enzyme (23,24). The crystal structure of TAO determined at 2.85 Å resolution (SI Appendix, Table S1) contains four monomers per asymmetric unit that associate to form homodimers ( Fig.…”

Section: Resultsmentioning

confidence: 99%

Structure of the trypanosome cyanide-insensitive alternative oxidase

Shiba¹,

Kido²,

Sakamoto³

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

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In addition to haem copper oxidases, all higher plants, some algae, yeasts, molds, metazoans, and pathogenic microorganisms such as Trypanosoma brucei contain an additional terminal oxidase, the cyanide-insensitive alternative oxidase (AOX). AOX is a diiron carboxylate protein that catalyzes the four-electron reduction of dioxygen to water by ubiquinol. In T. brucei, a parasite that causes human African sleeping sickness, AOX plays a critical role in the survival of the parasite in its bloodstream form. Because AOX is absent from mammals, this protein represents a unique and promising therapeutic target. Despite its bioenergetic and medical importance, however, structural features of any AOX are yet to be elucidated. Here we report crystal structures of the trypanosomal alternative oxidase in the absence and presence of ascofuranone derivatives. All structures reveal that the oxidase is a homodimer with the nonhaem diiron carboxylate active site buried within a four-helix bundle. Unusually, the active site is ligated solely by four glutamate residues in its oxidized inhibitor-free state; however, inhibitor binding induces the ligation of a histidine residue. A highly conserved Tyr220 is within 4 Å of the active site and is critical for catalytic activity. All structures also reveal that there are two hydrophobic cavities per monomer. Both inhibitors bind to one cavity within 4 Å and 5 Å of the active site and Tyr220, respectively. A second cavity interacts with the inhibitor-binding cavity at the diiron center. We suggest that both cavities bind ubiquinol and along with Tyr220 are required for the catalytic cycle for O 2 reduction. diiron protein | neglected tropical diseases | monotopic membrane protein | drug target | ubiquinol oxidase

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Section: Resultsmentioning

confidence: 99%

Structure of the trypanosome cyanide-insensitive alternative oxidase

Shiba¹,

Kido²,

Sakamoto³

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

156

209

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“…AOX was prepared exactly as detailed previously (Fedor et al., 2017, Jones et al., 2016), based on the work of Kido and coworkers (Kido et al., 2010). AOX was overexpressed in a hemA deficient derivative of Escherichia coli BL21(DE3) (FN102) (Nihei et al., 2003) from the plasmid pET15b- aox (Nihei et al., 2003) carrying an N-terminal Twin-Strep tag (Schmidt et al., 2013) and with the first 24 residues (mitochondrial targeting sequence) deleted.…”

Section: Methodsmentioning

confidence: 99%

Mitochondrial Supercomplexes Do Not Enhance Catalysis by Quinone Channeling

2018

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SummaryMitochondrial respiratory supercomplexes, comprising complexes I, III, and IV, are the minimal functional units of the electron transport chain. Assembling the individual complexes into supercomplexes may stabilize them, provide greater spatiotemporal control of respiration, or, controversially, confer kinetic advantages through the sequestration of local quinone and cytochrome c pools (substrate channeling). Here, we have incorporated an alternative quinol oxidase (AOX) into mammalian heart mitochondrial membranes to introduce a competing pathway for quinol oxidation and test for channeling. AOX substantially increases the rate of NADH oxidation by O2 without affecting the membrane integrity, the supercomplexes, or NADH-linked oxidative phosphorylation. Therefore, the quinol generated in supercomplexes by complex I is reoxidized more rapidly outside the supercomplex by AOX than inside the supercomplex by complex III. Our results demonstrate that quinone and quinol diffuse freely in and out of supercomplexes: substrate channeling does not occur and is not required to support respiration.

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“…While the presence of several different forms of NAD-G3PDH is relatively common in eukaryotes (15,16), two isoforms of FAD-G3PDH are, as far as we know, unique to trypanosomatids. To date, TAO and other components of the respiratory chain have been subjected to numerous investigations (8,12,14,27,29,(34)(35)(36)(37)(38)(39)(40)(41), while only two studies dealt with the function of mtG3PDH (12,14), and the role of putG3PDH remains totally unknown.…”

Section: Discussionmentioning

confidence: 99%

Characterization of Two Mitochondrial Flavin Adenine Dinucleotide-Dependent Glycerol-3-Phosphate Dehydrogenases in Trypanosoma brucei

et al. 2013

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dGlycerol-3-phosphate dehydrogenases (G3PDHs) constitute a shuttle that serves for regeneration of NAD ؉ reduced during glycolysis. This NAD-dependent enzyme is employed in glycolysis and produces glycerol-3-phosphate from dihydroxyacetone phosphate, while its flavin adenine dinucleotide (FAD)-dependent homologue catalyzes a reverse reaction coupled to the respiratory chain. Trypanosoma brucei possesses two FAD-dependent G3PDHs. While one of them (mitochondrial G3PDH [mtG3PDH]) has been attributed to the mitochondrion and seems to be directly involved in G3PDH shuttle reactions, the function of the other enzyme (putative G3PDH [putG3PDH]) remains unknown. In this work, we used RNA interference and protein overexpression and tagging to shed light on the relative contributions of both FAD-G3PDHs to overall cellular metabolism. Our results indicate that mtG3PDH is essential for the bloodstream stage of T. brucei, while in the procyclic stage the enzyme is dispensable. Expressed putG3PDH-V5 was localized to the mitochondrion, and the data obtained by digitonin permeabilization, Western blot analysis, and immunofluorescence indicate that putG3PDH is located within the mitochondrion.

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Purification and kinetic characterization of recombinant alternative oxidase from Trypanosoma brucei brucei

Cited by 50 publications

References 56 publications

Structure of the trypanosome cyanide-insensitive alternative oxidase

Structure of the trypanosome cyanide-insensitive alternative oxidase

Mitochondrial Supercomplexes Do Not Enhance Catalysis by Quinone Channeling

Characterization of Two Mitochondrial Flavin Adenine Dinucleotide-Dependent Glycerol-3-Phosphate Dehydrogenases in Trypanosoma brucei

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