Mutational analysis of the Trypanosoma vivax alternative oxidase: the E(X)6Y motif is conserved in both mitochondrial alternative oxidase and plastid terminal oxidase and is indispensable for enzyme activity.
Based on amino acid sequence similarity and the ability to catalyze the four-electron reduction of oxygen to water using a quinol substrate, mitochondrial alternative oxidase (AOX) and plastid terminal oxidase (PTOX) appear to be two closely related members of the membrane-bound diiron carboxylate group of proteins. In the current studies, we took advantage of the high activity of Trypanosoma vivax AOX (TvAOX) to examine the importance of the conserved Glu and the Tyr residues around the predicted third helix region of AOXs and PTOXs. We first compared the amino acid sequences of TvAOX with AOXs and PTOXs from various taxa and then performed alanine-scanning mutagenesis of TvAOX between amino acids Y(199) and Y(247). We found that the ubiquinol oxidase activity of TvAOX is completely lost in the E214A mutant, whereas mutants E215A and E216A retained more than 30% of the wild-type activity. Among the Tyr mutants, a complete loss of activity was also observed for the Y221A mutant, whereas the activities were equivalent to wild-type for the Y199A, Y212A, and Y247A mutants. Finally, residues Glu(214) and Tyr(221) were found to be strictly conserved among AOXs and PTOXs. Based on these findings, it appears that AOXs and PTOXs are a novel subclass of diiron carboxylate proteins that require the conserved motif E(X)(6)Y for enzyme activity.
Nakamura
Kosuke
K
Department of Biomedical Chemistry, Graduate School of Medicine, The University of Tokyo, Tokyo 113-0033, Japan.
Sakamoto
Kimitoshi
K
Kido
Yasutoshi
Y
Fujimoto
Yoko
Y
Suzuki
Takashi
T
Suzuki
Mitsuko
M
Yabu
Yoshisada
Y
Ohta
Nobuo
N
Tsuda
Akiko
A
Onuma
Misao
M
Kita
Kiyoshi
K
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D017124:Conserved Sequence; D004252:DNA Mutational Analysis / Q000379:methods*; D004789:Enzyme Activation; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D008928:Mitochondria / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010088:Oxidoreductases / Q000032:analysis / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D018087:Plastids / Q000201:enzymology*; D011994:Recombinant Proteins / Q000032:analysis / Q000737:chemistry / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013329:Structure-Activity Relationship
D011994:Recombinant Proteins; D010088:Oxidoreductases; C020094:duroquinol oxidase