A novel functional element in the N-terminal region of Arum concinnatum alternative oxidase is indispensable for catalytic activity of the enzyme in HeLa cells.
Alternative oxidase (AOX) is a quinol-oxygen oxidoreductase, which is known to possess a dicarboxylate diiron reaction center held in structurally postulated alpha-helical bundle. However, little is known about the structural or functional features of its N-terminal region in any organism, with the exception of a regulatory cysteine residue (CysI) in angiosperm plants. Here, we show that transcripts of two AOX1 isozymes (AcoAOX1a and AcoAOX1b) are coexpressed in thermogenic appendices of Arum concinnatum, while their enzymatic activities seem to be distinct. Namely, AcoAOX1a, an abundantly expressed transcript in vivo, shows an apparent cyanide-insensitive and n-propyl gallate-sensitive respiration during ectopic expression of the protein in HeLa cells, whereas AcoAOX1b exhibits a lower transcript expression, and appears to be totally inactive as AOX at the protein level. Our functional analyses further reveal that an E83K substitution in AcoAOX1b, which is located far upstream of CysI in the N-terminal region, is the cause of this loss of function. These results suggest the presence of a naturally occurring inactive AOX homologue in thermogenic plants. Accordingly, our results further imply that the N-terminal region of the AOX protein functionally contributes to the dynamic activities of respiratory control within the mitochondria.
Kakizaki
Yusuke
Y
Cryobiofrontier Research Center, Faculty of Agriculture, Iwate University 3-18-8 Ueda, Morioka, Iwate 020-8550, Japan.
Seymour
Roger S
RS
Ito
Kikukatsu
K
D031058:Arum / Q000201:enzymology*; D002384:Catalysis; D017931:DNA Primers; D015342:DNA Probes; D006367:HeLa Cells / Q000201:enzymology*; D006801:Humans; D007527:Isoenzymes / Q000235:genetics / Q000378:metabolism; D024101:Mitochondrial Proteins; D010088:Oxidoreductases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D010101:Oxygen Consumption; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D010940:Plant Proteins; D018749:RNA, Plant / Q000235:genetics; D011994:Recombinant Proteins / Q000378:metabolism; D012260:Ribonucleases; D013816:Thermodynamics; D014158:Transcription, Genetic; D014162:Transfection; D000099188:Alternative Oxidase
D017931:DNA Primers; D015342:DNA Probes; D007527:Isoenzymes; D024101:Mitochondrial Proteins; D010088:Oxidoreductases; D010446:Peptide Fragments; D010940:Plant Proteins; D018749:RNA, Plant; D011994:Recombinant Proteins; D012260:Ribonucleases; D000099188:Alternative Oxidase