X-ray Structure of β-Carbonic Anhydrase from the Red Alga,Porphyridium purpureum, Reveals a Novel Catalytic Site for CO2 Hydration

Abstract

The carbonic anhydrases (CAs) fall into three evolutionarily distinct families designated ␣-, ␤-, and ␥-CAs based on their primary structure. ␤-CAs are present in higher plants, algae, and prokaryotes, and are involved in inorganic carbon utilization. Here, we describe the novel x-ray structure of ␤-CA from the red alga, Porphyridium purpureum, at 2.2-Å resolution using intrinsic zinc multiwavelength anomalous diffraction phasing. The CA monomer is composed of two internally repeating structures, being folded as a pair of fundamentally equivalent motifs of an ␣/␤ domain and three projecting ␣-helices. The motif is obviously distinct from that of either ␣-or ␥-CAs. This homodimeric CA appears like a tetramer with a pseudo 222 symmetry. The active site zinc is coordinated by a Cys-Asp-His-Cys tetrad that is strictly conserved among the ␤-CAs. No water molecule is found in a zinc-liganding radius, indicating that the zinc-hydroxide mechanism in ␣-CAs, and possibly in ␥-CAs, is not directly applicable to the case in ␤-CAs. Zinc coordination environments of the CAs provide an interesting example of the convergent evolution of distinct catalytic sites required for the same CO 2 hydration reaction.Carbonic anhydrase (CA, 1 EC 4.2.1.1) is a zinc-containing enzyme which catalyzes the reversible hydration of CO 2 . CA is ubiquitously distributed in nature and is involved in fundamental biological processes such as photosynthesis, respiration, pH homeostasis, and ion transport (1, 2). Based on sequence homologies, CAs are classified into three evolutionarily distinct groups, designated ␣-, ␤-, and ␥-CAs (3). ␣-CAs are found in mammals, algae, and prokaryotes, ␤-CAs in higher plants, algae, and prokaryotes, and ␥-CA has been identified in the archaeon, Methanosarcina thermophila, although gene homologies have been recently found in higher plants and prokaryotes (4).The x-ray structures of ␣-CAs from several mammals (5, 6) have revealed a common overall structure dominated by ␤-sheets. The catalytically active zinc is liganded by three histidine residues with a hydroxide or a water molecule as a fourth ligand, giving a tetrahedral coordination geometry. For the CO 2 hydration reaction, the zinc-bound hydroxide initiates a nucleophilic attack on the substrate CO 2 to form zinc-bound HCO 3 Ϫ which is then displaced by a water molecule (7). The regeneration of zinc-bound hydroxide requires an intramolecular proton shuttle from the zinc-bound water to the bulk solvent for each cycle of catalysis. The proton transfer is ratelimiting in the catalysis (8).The x-ray structure of ␥-CA from M. thermophila has been reported (4). This is a trimeric CA with a peculiar left-handed ␤-helical folding motif. The active site zinc is located at subunit interfaces and coordinated by two histidines from one subunit and one histidine from a neighboring one. In addition, at an electron dense region which occupies the fourth coordination site, a putative water molecule is recognized. Although the polypeptide assembly forming the active site of the ...

Carbonic anhydrases in plants and algae

“…The pea CA is an octamer in which dimers form tetramers that form octamers.Active sites are at the subunit interfaces and as suggested by EXAFS, mutagenesis and elemental analysis (Rowlett et al 1994;Bracey et al 1994), the zinc is bound by two cysteines and one histidine. In the algal CA an aspartate rather than water occupies the fourth co-ordination position (Mitsuhashi et al 2000). The algal CA was crystallized in the absence of substrate (or inhibitor) whereas the pea enzyme was crystallized in the presence of acetate.…”

Structural and Kinetic Characterization of an Archaeal β-Class Carbonic Anhydrase

“…In stark contrast to the mainly ␤-sheet structures of ␣-and ␥-class carbonic anhydrases, CD analysis of Cab and the S. oleracea enzyme suggests a predominantly ␣-helical structure. These prokaryotic and eukaryotic ␤-class carbonic anhydrases are from organisms at the phylogenetic extremes, suggesting this secondary structure feature is common to all ␤-class enzymes; indeed, the crystal structures of the P. sativum and P. purpureum ␤-class enzymes reveal a predominance of ␣-helical structure (35,41). The content and arrangement of the predicted secondary structure elements for Cab are similar to those of the other ␤-class carbonic anhydrases (Fig.…”

“…5). The recently solved crystal structures of the P. sativum and P. purpureum ␤-class carbonic anhydrase confirm that the two conserved cysteines and the conserved histidine are ligands to the active site zinc (35,41). The second oxygen/nitrogen ligand would be expected to be a water molecule; however, the fourth ligand of the recently solved P. purpureum crystal structure is a conserved aspartate corresponding to Asp-34 of Cab.…”

“…The dimeric P. purpureum enzyme, in which each monomer is composed of two internally repeated structures each having an active site (Fig. 5), appears as a tetramer with a pseudo 222 symmetry (41). Why alterations to the invariant Cys-269 and Cys-272 of the dicotyledenous plant enzymes convert the octamer into a tetramer is unclear.…”

“…Even though the ␣-and ␥-classes are notably different in both their tertiary and quaternary structures, both classes contain a catalytically essential zinc ion coordinated by three histidine residues. Recently, the structures of the ␤-class carbonic anhydrases from both the dicotyledenous plant Pisum sativum (pea) (35) and the red alga Porphyridium purpureum (41) have been solved. Both the P. sativum homo-octamer and the P. purpureum homodimer exhibit a predominantly ␣-helical secondary structure.…”