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

Abstract

In recent years, members of the β class of CAs (carbonic anhydrases) have been shown to complement ΔNCE103, a yeast strain unable to grow under aerobic conditions. The activity required for complementation of ΔNCE103 by tobacco chloroplast CA was studied by site-directed mutagenesis. E196A (Glu196→Ala), a mutated tobacco CA with low levels of CA activity, complemented ΔNCE103. To determine whether restoration of ΔNCE103 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 α CA structurally unrelated to the β class of CAs to which the tobacco protein belongs. Human CAII complemented ΔNCE103, strongly arguing that CA activity is responsible for the complementation of ΔNCE103. Consistent with this conclusion, recombinant NCE103 synthesized in Escherichia coli shows CA activity, and ΔNCE103 expressing the tobacco chloroplast CA exhibits the same sensitivity to H2O2 as the wild-type strain.