Complementation of the yeast deletion mutant DeltaNCE103 by members of the beta class of carbonic anhydrases is dependent on carbonic anhydrase activity rather than on antioxidant activity.
In recent years, members of the beta class of CAs (carbonic anhydrases) have been shown to complement Delta NCE103, a yeast strain unable to grow under aerobic conditions. The activity required for complementation of Delta NCE103 by tobacco chloroplast CA was studied by site-directed mutagenesis. E196A (Glu196-->Ala), a mutated tobacco CA with low levels of CA activity, complemented Delta NCE103. To determine whether restoration of Delta NCE103 was due to residual levels of CA activity or whether it was related to previously proposed antioxidant activity of CAs [Götz, Gnann and Zimmermann (1999) Yeast 15, 855-864], additional complementation analysis was performed using human CAII, an alpha CA structurally unrelated to the beta class of CAs to which the tobacco protein belongs. Human CAII complemented Delta NCE103, strongly arguing that CA activity is responsible for the complementation of Delta NCE103. Consistent with this conclusion, recombinant NCE103 synthesized in Escherichia coli shows CA activity, and Delta NCE103 expressing the tobacco chloroplast CA exhibits the same sensitivity to H2O2 as the wild-type strain.
Clark
Daniel
D
Boyce Thompson Institute for Plant Research, Tower Road, Ithaca, NY 14853, USA.
Rowlett
Roger S
RS
Coleman
John R
JR
Klessig
Daniel F
DF
D000595:Amino Acid Sequence; D000975:Antioxidants / Q000378:metabolism*; D024402:Carbonic Anhydrase II / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D002256:Carbonic Anhydrases / Q000737:chemistry / Q000145:classification* / Q000235:genetics / Q000378:metabolism*; D002736:Chloroplasts / Q000201:enzymology; D017353:Gene Deletion; D005816:Genetic Complementation Test; D006801:Humans; D006861:Hydrogen Peroxide / Q000378:metabolism / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed / Q000235:genetics; D016877:Oxidants / Q000378:metabolism / Q000494:pharmacology; D018384:Oxidative Stress / Q000187:drug effects; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D014026:Nicotiana / Q000166:cytology / Q000201:enzymology / Q000235:genetics
D000975:Antioxidants; D016877:Oxidants; D006861:Hydrogen Peroxide; D024402:Carbonic Anhydrase II; D002256:Carbonic Anhydrases