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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 ...

RIKEN beamline I (BL45XU) is an undulator beamline with two branches. One is for protein crystallography (PX) and the other is for small‐angle x‐ray scattering (SAXS). The beam is split into the two branches by a diamond monochromator so that two experiments can be done simultaneously [Yamamoto et al. (1995) Rev. Sci. Instrum.66, 1833‐1835]. The SAXS branch was designed for studying the weak interaction of proteins or subunits of fibrous or protein solutions especially using hydrostatic pressure. The optics makes use of the good parallelism of the undulator beam in order to reduce parasitic scattering. The beamline consists of a double crystal monochromator and a K‐B type focusing mirror system. In order to cope with the high flux of the beam, an x‐ray image intensifier (Hamamatsu Photonics, V5445P) with a cooled CCD camera (C4880‐82) was used. As a result, decreases in both collection time and sample amount were realized in standard static experiments. These improvements will greatly facilitate SAXS experiments under high pressure.