Alternative Oxidase (AtAox) c78s Mutant Expression at Escherichia coli (SASX41DB)

• Main Author: Ira Djajanegara
• Format: Article
• Language: English
• Published: Universitas Gadjah Mada, Yogyakarta 2015-11-01
• Series: Indonesian Journal of Biotechnology
• Online Access: http://journal.ugm.ac.id/ijbiotech/article/view/7766
• ISSN: 0853-8654; 2089-2241

Alternative oxidase (AOX) is the terminal oxidase operating in the mitochondrial electron transport chain. The<br />enzyme is activated by organic acid such as pyruvate and by reduction process. Based on sequences alignment of<br />alternative oxidase gene (Aox) found in several organisms, there are 2 conserved cysteine residues. In order to<br />investigate the importance of those cysteine residues on the activity of AOX, mutation at cysteine residue number 78<br />of Aox gene isolated from Arabidopsis thaliana (AtAox) was conducted. Cysteine at position number 78 was changed<br />into serine and the c78s mutant was expressed in Escherichia coli strain SASX41DB. This particular E. coli strain is<br />unable to grow aerobically unless transformed with Arabidopsis Aox gene (AtAox). Expression studies on c78s<br />mutant showed that this mutant cannot be oxididized and can not be activated by pyruvic acid. This mutant is<br />acivated by succinate instead of pyruvate. Mutation at cysteine closer to the N residue is affecting both organic acid<br />and redox activation. Therefore, it is concluded that cysteine residue closer to the N residue is the site for both<br />activation by pyruvate as well as activation by reduction process.<br />Keywords : Alternative oxidase, site-directed mutation, SASx41DB, cysteine residues