A single amino acid change in the plant alternative oxidase alters the specificity of organic acid activation.
The alternative oxidase is a quinol oxidase of the respiratory chain of plants and some fungi and protists. Its activity is regulated by redox-sensitive disulphide bond formation between neighbouring subunits and direct interaction with certain alpha-ketoacids. To investigate these regulatory mechanisms, we undertook site-directed mutagenesis of soybean and Arabidopsis alternative oxidase cDNAs, and expressed them in tobacco plants and Escherichia coli, respectively. The homologous C99 and C127 residues of GmAOX3 and AtAOX1a, respectively, were changed to serine. In the plant system, this substitution prevented oxidative inactivation of alternative oxidase and rendered the protein insensitive to pyruvate activation, in agreement with the recent results from other laboratories [Rhoads et al. (1998) J. Biol. Chem. 273, 30750-30756; Vanlerberghe et al. (1998) Plant Cell 10, 1551-1560]. However, the mutated protein is instead activated specifically by succinate. Measurements of AtAOX1a activity in bacterial membranes lacking succinate dehydrogenase confirmed that the stimulation of the mutant protein's activity by succinate did not involve its metabolism. Examples of alternative oxidase proteins with the C to S substitution occur in nature and these oxidases are expected to be activated under most conditions in vivo, with implications for the efficiency of respiration in the tissues which express them.
Djajanegara
I
I
Division of Biochemistry and Molecular Biology, Faculty of Science, Australian National University, Canberra, ACT.
Holtzapffel
R
R
Finnegan
P M
PM
Hoefnagel
M H
MH
Berthold
D A
DA
Wiskich
J T
JT
Day
D A
DA
D019943:Amino Acid Substitution; D017360:Arabidopsis / Q000201:enzymology* / Q000235:genetics; D004789:Enzyme Activation / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D015971:Gene Expression Regulation, Enzymologic; D018506:Gene Expression Regulation, Plant; D024101:Mitochondrial Proteins; D010088:Oxidoreductases / Q000235:genetics* / Q000378:metabolism; D010940:Plant Proteins / Q000235:genetics / Q000378:metabolism; D010947:Plants, Toxic; D014026:Nicotiana / Q000235:genetics; D000099188:Alternative Oxidase
D024101:Mitochondrial Proteins; D010088:Oxidoreductases; D010940:Plant Proteins; D000099188:Alternative Oxidase