Conserved active site sequences in Arabidopsis plastid terminal oxidase (PTOX): in vitro and in planta mutagenesis studies.
The plastid terminal oxidase (PTOX) is distantly related to the mitochondrial alternative oxidase (AOX). Both are members of the diiron carboxylate quinol oxidase (DOX) class of proteins. PTOX and AOX contain 20 highly conserved amino acids, six of which are Fe-binding ligands. We have previously used in vitro and in planta activity assays to examine the functional importance of the Fe-binding sites. In this report, we conduct alanine-scanning mutagenesis on the 14 other conserved sites using our in vitro and in planta assay procedures. We found that the 14 sites fall into three classes: (i) Ala-139, Pro-142, Glu-171, Asn-174, Leu-179, Pro-216, Ala-230, Asp-287, and Arg-293 are dispensable for activity; (ii) Tyr-234 and Asp-295 are essential for activity; and (iii) Leu-135, His-151, and Tyr-212 are important but not essential for activity. Our data are consistent with the proposed role of some of these residues in active site conformation, substrate binding, and/or catalysis. Titration experiments showed that down-regulation of PTOX to approximately 3% of wild-type levels did not compromise plant growth, at least under ambient growth conditions. This suggests that PTOX is normally in excess, especially early in thylakoid membrane biogenesis.
Fu
Aigen
A
Department of Genetics, Development and Cell Biology, Iowa State University, Ames, Iowa 50011, USA.
Aluru
Maneesha
M
Rodermel
Steven R
SR
D017360:Arabidopsis / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development; D029681:Arabidopsis Proteins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D015153:Blotting, Western; D020134:Catalytic Domain / Q000235:genetics; D024101:Mitochondrial Proteins; D016297:Mutagenesis, Site-Directed; D010088:Oxidoreductases / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D010940:Plant Proteins; D030821:Plants, Genetically Modified / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development; D011485:Protein Binding; D000099188:Alternative Oxidase
D029681:Arabidopsis Proteins; D024101:Mitochondrial Proteins; D010088:Oxidoreductases; D010940:Plant Proteins; D000099188:Alternative Oxidase; C405742:IM protein, Arabidopsis