Characterization of pea chloroplastic carbonic anhydrase. Expression in Escherichia coli and site-directed mutagenesis.
A cDNA encoding the mature, chloroplast-localized carbonic anhydrase in pea has been expressed in E. coli. The enzyme is fully active and yields of up to 20% of the total soluble protein can be obtained from the bacteria. This expression system was used to monitor the effects of site-directed mutagenesis of seven residues found within conserved regions in the pea carbonic anhydrase amino acid sequence. The effects of these modifications are discussed with respect to the potential of various amino acids to act as sites for zinc coordination or intramolecular proton shuttles.
Dept. of Botany, University of Toronto, Ontario, Canada.
N J
NJ
Provart
N
N
Majeau
J R
JR
Coleman
D000595:Amino Acid Sequence; D002256:Carbonic Anhydrases / Q000235:genetics / Q000378:metabolism*; D002736:Chloroplasts / Q000201:enzymology*; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli; D007887:Fabaceae / Q000201:enzymology*; D015971:Gene Expression Regulation, Enzymologic; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010946:Plants, Medicinal; D012995:Solubility; D013329:Structure-Activity Relationship
D002256:Carbonic Anhydrases