title: Purification and characterisation of recombinant DNA encoding the alternative oxidase from Sauromatum guttatum.
abstract: The alternative oxidase (AOX) is a non-protonmotive ubiquinol oxidase that is found in mitochondria of all higher plants studied to date. Structural and functional characterisation of this important but enigmatic plant diiron protein has been hampered by an inability to obtain sufficient native protein from plant sources. In the present study recombinant SgAOX (rSgAOX), overexpressed in a ΔhemA-deficient Escherichia coli strain (FN102), was solubilized from E. coli membranes and purified to homogeneity in a stable and highly active form. The kinetics of ubiquinol-1 oxidation by purified rSgAOX showed typical Michaelis-Menten kinetics (K(m) of 332 μM and Vmax of 30 μmol(-1) min(-1) mg(-1)), a turnover number 20 μmol s(-1) and a remarkable stability. The enzyme was potently inhibited not only by conventional inhibitors such as SHAM and n-propyl gallate but also by the potent TAO inhibitors ascofuranone, an ascofuranone-derivative colletochlorin B and the cytochrome bc1 inhibitor ascochlorin. Circular dichroism studies revealed that AOX was approximately 50% α-helical and furthermore such studies revealed that rSgAOX and rTAO partially retained the helical absorbance signal even at 90 °C (58% and 64% respectively) indicating a high conformational stability. It is anticipated that highly purified and active AOX and its mutants will facilitate investigations into the structure and reaction mechanisms of AOXs through the provision of large amounts of purified protein for crystallography and contribute to further progress of the study on this important plant terminal oxidase.
authors.affiliation: Biochemistry and Molecular Sciences, School of Life Sciences, University of Sussex, Falmer, Brighton BN1 9QG, United Kingdom.
authors.forename: Catherine
authors.initials: C
authors.lastname: Elliott
authors.affiliation: Biochemistry and Molecular Sciences, School of Life Sciences, University of Sussex, Falmer, Brighton BN1 9QG, United Kingdom.
authors.forename: Luke
authors.initials: L
authors.lastname: Young
authors.affiliation: Biochemistry and Molecular Sciences, School of Life Sciences, University of Sussex, Falmer, Brighton BN1 9QG, United Kingdom.
authors.forename: Ben
authors.initials: B
authors.lastname: May
authors.affiliation: Biochemistry and Molecular Sciences, School of Life Sciences, University of Sussex, Falmer, Brighton BN1 9QG, United Kingdom.
authors.forename: Julia
authors.initials: J
authors.lastname: Shearman
authors.affiliation: Biochemistry and Molecular Sciences, School of Life Sciences, University of Sussex, Falmer, Brighton BN1 9QG, United Kingdom.
authors.forename: Mary S
authors.initials: MS
authors.lastname: Albury
authors.affiliation: Department of Biomedical Chemistry, Graduate School of Medicine, The University of Tokyo, Tokyo 113-0033, Japan.
authors.forename: Yasutoshi
authors.initials: Y
authors.lastname: Kido
authors.affiliation: Department of Biomedical Chemistry, Graduate School of Medicine, The University of Tokyo, Tokyo 113-0033, Japan.
authors.forename: Kiyoshi
authors.initials: K
authors.lastname: Kita
authors.affiliation: Biochemistry and Molecular Sciences, School of Life Sciences, University of Sussex, Falmer, Brighton BN1 9QG, United Kingdom. Electronic address: a.l.moore@sussex.ac.uk.
authors.forename: Anthony L
authors.initials: AL
authors.lastname: Moore
mesh_terms: D029064:Araceae / Q000201:enzymology* / Q000235:genetics; D002942:Circular Dichroism; D003001:Cloning, Molecular; D004791:Enzyme Inhibitors / Q000032:analysis; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D006358:Hot Temperature; D007700:Kinetics; D024101:Mitochondrial Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D010084:Oxidation-Reduction; D010088:Oxidoreductases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D010940:Plant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D011487:Protein Conformation; D055550:Protein Stability; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D014451:Ubiquinone / Q000031:analogs & derivatives / Q000378:metabolism; D000099188:Alternative Oxidase
chemical_list: D004791:Enzyme Inhibitors; D024101:Mitochondrial Proteins; D010088:Oxidoreductases; D010940:Plant Proteins; D011994:Recombinant Proteins; D014451:Ubiquinone; D000099188:Alternative Oxidase; C003741:ubiquinol
keywords: Alternative oxidase; Ascofuranone; Escherichia coli membranes; Protein purification; Quinol oxidase; Site-directed mutagenesis