Purification and characterisation of recombinant DNA encoding the alternative oxidase from Sauromatum guttatum

Elliott, Kate, Young, Luke, May, Ben, Shearman, Julia, Albury, Mary S, Kido, Yasutoshi, Kita, Kiyoshi and Moore, Anthony L (2014) Purification and characterisation of recombinant DNA encoding the alternative oxidase from Sauromatum guttatum. Mitochondrion, 19 (Part B). pp. 261-268. ISSN 1567-7249

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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 (Km of 332 μMand 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.

Item Type: Article
Schools and Departments: School of Life Sciences > Biochemistry
Subjects: Q Science
Q Science > Q Science (General) > Q0179.9 Research
Q Science > Q Science (General)
Depositing User: Anthony Moore
Date Deposited: 11 Dec 2017 15:37
Last Modified: 11 Dec 2017 15:37
URI: http://sro.sussex.ac.uk/id/eprint/71981

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