Crystal structure of E. coli β–carbonic anhydrase, an enzyme with an unusual pH–dependent activity

• Published in: Protein science Vol. 10; no. 5; pp. 911 - 922
• Main Authors: Cronk, Jeff D., Endrizzi, James A., Cronk, Michelle R., O'neill, Jason W., Zhang, Kam Y.J.
• Format: Journal Article
• Language: English
• Published: Bristol Cold Spring Harbor Laboratory Press 01.05.2001
• Subjects: Amino Acid Sequence; Binding Sites; CA, carbonic anhydrase; Carbonic anhydrase; Carbonic Anhydrases - chemistry; Carbonic Anhydrases - isolation & purification; Carbonic Anhydrases - metabolism; crystal structure; Crystallography, X-Ray; ECCA, Escherichia coli β‐carbonic anhydrase; Escherichia coli - enzymology; EXAFS, extended X‐ray absorption fine structure spectroscopy; Gene Deletion; Genetic Complementation Test; Hydrogen-Ion Concentration; MAD, multiwavelength anomalous dispersion; metalloenzyme; Models, Molecular; Molecular Sequence Data; pH‐dependent activity; PPCA, Porphyridium purpureum β‐carbonic anhydrase; Protein Folding; Protein Structure, Quaternary; Protein Structure, Secondary; PSCA, Pisum sativum β‐carbonic anhydrase; Saccharomyces cerevisiae - enzymology; Saccharomyces cerevisiae - genetics; Saccharomyces cerevisiae - growth & development; Structure-Activity Relationship; Zinc - metabolism; zinc coordination
• ISSN: 0961-8368; 1469-896X
• DOI: 10.1110/ps.46301

Carbonic anhydrases fall into three distinct evolutionary and structural classes: α, β, and γ. The β‐class carbonic anhydrases (β‐CAs) are widely distributed among higher plants, simple eukaryotes, eubacteria, and archaea. We have determined the crystal structure of ECCA, a β‐CA from Escherichia coli, to a resolution of 2.0 Å. In agreement with the structure of the β‐CA from the chloroplast of the red alga Porphyridium purpureum, the active‐site zinc in ECCA is tetrahedrally coordinated by the side chains of four conserved residues. These results confirm the observation of a unique pattern of zinc ligation in at least some β‐CAs. The absence of a water molecule in the inner coordination sphere is inconsistent with known mechanisms of CA activity. ECCA activity is highly pH‐dependent in the physiological range, and its expression in yeast complements an oxygen‐sensitive phenotype displayed by a β‐CA‐deletion strain. The structural and biochemical characterizations of ECCA presented here and the comparisons with other β‐CA structures suggest that ECCA can adopt two distinct conformations displaying widely divergent catalytic rates.