Spinach Carbonic Anhydrase: Primary Sequence, Transport Into Chloroplasts and Expression in Escherichia Coli.

Carbonic anhydrase (CA) catalyzes the reversible hydration of C02. Plant carbonic anhydrases are structurally distinct from those found in animals. In C3 plants, the enzyme is located in chloroplasts. To date, little structural information on the enzyme from plants has been available. In this study the full-length cDNA for the spinach chloroplast CA was sequenced. The cDNA contained 1,156 base pairs. The open reading frame encoded a protein of 34,569 daltons. Comparison of the N-terminal sequence of this protein with that of other chloroplast precursors indicated that CA is synthesized as a precursor. The transit peptide likely contains about 60 amino acids. Indirect immunoprecipitation of translation products synthesized using pea poly A RNA indicated that the pea precursor is approximately 36,000 daltons. Incubation of either the pea or spinach precursor with isolated intact chloroplasts resulted in import of the precursor and cleavage to a polypeptide of about 30,000 daltons. Various spinach CAs containing deletions ranging from 34 to 78 amino acids were expressed in IL. coli. under the control of the trc promotor. Each of the CAs assembled to yield the enzymatically active