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

Abstract

The ␤-class carbonic anhydrase from the archaeon Methanobacterium thermoautotrophicum (Cab) was structurally and kinetically characterized. Analytical ultracentrifugation experiments show that Cab is a tetramer. Circular dichroism studies of Cab and the Spinacia oleracea (spinach) ␤-class carbonic anhydrase indicate that the secondary structure of the ␤-class enzymes is predominantly ␣-helical, unlike that of the ␣-or ␥-class enzymes. Extended X-ray absorption fine structure results indicate the active zinc site of Cab is coordinated by two sulfur and two O/N ligands, with the possibility that one of the O/N ligands is derived from histidine and the other from water. Both the steady-state parameters k cat and k cat /K m for CO 2 hydration are pH dependent. The steady-state parameter k cat is buffer-dependent in a saturable manner at both pH 8.5 and 6.5, and the analysis suggested a ping-pong mechanism in which buffer is the second substrate. At saturating buffer conditions and pH 8.5, k cat is 2.1-fold higher in H 2 O than in D 2 O, consistent with an intramolecular proton transfer step being rate contributing. The steady-state parameter k cat /K m is not dependent on buffer, and no solvent hydrogen isotope effect was observed. The results suggest a zinc hydroxide mechanism for Cab. The overall results indicate that prokaryotic ␤-class carbonic anhydrases have fundamental characteristics similar to the eukaryotic ␤-class enzymes and firmly establish that the ␣-, ␤-, and ␥-classes are convergently evolved enzymes that, although structurally distinct, are functionally equivalent.The thermophilic archaeon Methanobacterium thermoautotrophicum obtains energy for growth by the reduction of CO 2 to CH 4 and is also an obligate chemolithoautotroph; thus, this organism has a high demand for CO 2 . Carbonic anhydrase, a zinc-containing enzyme catalyzing the reversible hydration of carbon dioxide (equation 1)is expected to play an important role in the growth of M. thermoautotrophicum and may have several functions, including transporting HCO 3 Ϫ into the cell and providing CO 2 or HCO 3 Ϫ to enzymes that utilize these substrates. Based on sequence comparisons, carbonic anhydrases belong to three genetically distinct classes (␣, ␤, and ␥) which appear to have independent origins (24). The most extensively studied enzymes are those from the ␣-class, which is composed primarily of mammalian carbonic anhydrases, but also includes enzymes from the green alga Chlamydomonas reinhardtii (19,20) and the prokaryote Neisseria gonorrhoeae (13). The ␤-class enzymes are abundant in C 3 and C 4 monocotyledenous and dicotyledenous plants and green unicellular algae (24, 43), where they are essential for photosynthetic CO 2 fixation (6). The most recently identified class of carbonic anhydrase, the ␥-class (24), is represented by the prototype Cam from the archaeon Methanosarcina thermophila (2). Even though sequences encoding putative ␥-class carbonic anhydrases have been found in prokaryotes from both the Bacteria and Archaea domains (2...

Roles of the Conserved Aspartate and Arginine in the Catalytic Mechanism of an Archaeal β-Class Carbonic Anhydrase

“…Of the ␤-class carbonic anhydrases, only two plant-type ␤-class carbonic anhydrases and one cab-type ␤-class carbonic anhydrase have been kinetically characterized (14,15,28,31). Even before the structure of any ␤-class enzyme had been determined, Asp-34 and Arg-36 of Cab were obvious targets for site-specific replacement to determine their roles in catalysis.…”

“…The wild-type and variant carbonic anhydrases were heterologously produced in Escherichia coli BL21(DE3) (Novagen, Inc.) and purified as previously described (31,32). Enzyme activity during purification was measured at room temperature by using a modification of the electrometric method of Wilbur and Anderson (41).…”

“…The catalytically active group in this mechanistic model is the zinc-bound water, which ionizes to a metal-bound hydroxyl which attacks CO 2 . Despite considerable structural differences in the active sites of these enzymes, the catalytic mechanisms of both ␥-and ␤-class enzymes resemble those of human ␣-class HCA II (1,14,15,28,31). The overall enzyme-catalyzed reaction occurs in two mechanistically distinct steps (where E ϭ enzyme and B ϭ buffer): …”

Biochemistry and Physiology of the β Class Carbonic Anhydrase (Cpb) from Clostridium perfringens Strain 13

“…Although commercially available bovine ␣ class CA showed an esterase activity of ϳ37 mol of p-nitrophenylacetate hydrolyzed min Ϫ1 mol Ϫ1 of enzyme, the C. perfringens CA (Cpb) had no detectable esterase activity (Ͻ0.05 mol of p-nitrophenylacetate hydrolyzed min Ϫ1 mol Ϫ1 of tetramer), also a property of Cab from M. thermautotrophicus (16,17).…”

“…The ␤ class CAs of photosynthetic species function to concentrate atmospheric CO 2 , whereas it is proposed that nonphotosynthetic species utilize ␤ class CAs to retain intracellular CO 2 by conversion to bicarbonate for anaplerotic carboxylation reactions (11)(12)(13)(14). The only characterized ␤ class CA from the domain Archaea (Cab) is that from Methanothermobacter thermoautotrophicus (formerly Methanobacterium thermoautotrophicum strain ⌬H) (15)(16)(17)(18), a microbe that obtains energy for growth by reducing CO 2 to methane. It is proposed that Cab functions to concentrate CO 2 for both methanogenesis and anaplerotic carboxylation reactions.…”