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

Abstract

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