A Closer Look at the Active Site of γ-Class Carbonic Anhydrases:  High-Resolution Crystallographic Studies of the Carbonic Anhydrase from Methanosarcina thermophila^,

Abstract

The prototype of the gamma-class of carbonic anhydrase has been characterized from the methanogenic archaeon Methanosarcina thermophila. Previously reported kinetic studies of the gamma-class carbonic anhydrase are consistent with this enzyme having a reaction mechanism similar to that of the mammalian alpha-class carbonic anhydrase. However, the overall folds of these two enzymes are dissimilar, and apart from the zinc-coordinating histidines, the active site residues bear little resemblance to one another. The crystal structures of zinc-containing and cobalt-substituted gamma-class carbonic anhydrases from M. thermophila are reported here between 1.46 and 1.95 A resolution in the unbound form and cocrystallized with either SO(4)(2)(-) or HCO(3)(-). Relative to the tetrahedral coordination geometry seen at the active site in the alpha-class of carbonic anhydrases, the active site of the gamma-class enzyme contains additional metal-bound water ligands, so the overall coordination geometry is trigonal bipyramidal for the zinc-containing enzyme and octahedral for the cobalt-substituted enzyme. Ligands bound to the active site all make contacts with the side chain of Glu 62 in manners that suggest the side chain is likely protonated. In the uncomplexed zinc-containing enzyme, the side chains of Glu 62 and Glu 84 appear to share a proton; additionally, Glu 84 exhibits multiple conformations. This suggests that Glu 84 may act as a proton shuttle, which is an important aspect of the reaction mechanism of alpha-class carbonic anhydrases. A hydrophobic pocket on the surface of the enzyme may participate in the trapping of CO(2) at the active site. On the basis of the coordination geometry at the active site, ligand binding modes, the behavior of the side chains of Glu 62 and Glu 84, and analogies to the well-characterized alpha-class of carbonic anhydrases, a more-defined reaction mechanism is proposed for the gamma-class of carbonic anhydrases.

A Multiprotein Bicarbonate Dehydration Complex Essential to Carboxysome Function in Cyanobacteria

“…Structural and amino acid sequence comparisons indicated that all of the conserved Zncoordinating histidine residues and other residues identified as important for the binding of HCO 3 -and the formation of the Cam trimer are conserved in CcmM73 (41). These residues mapped to similar spatial locations when computer-generated models of the N-terminal region of CcmM73 were superimposed (http://wishart.biology.ualberta.ca/SuperPose/) (23) onto the experimentally determined crystal structure of Cam (14,19), with root-mean-square deviations ranging from 1.4 to 4 Å. Thus, the bioinformatics analysis encourages the view that CcmM73 trimers may retain HCO 3 -/CO 2 binding capability, although the experimental evidence indicates that CcmM73 is apparently unable to catalyze HCO 3 -dehydration (Fig.…”

The Cytoplasmic Carbonic Anhydrases βCA2 and βCA4 Are Required for Optimal Plant Growth at Low CO2

“…The active unit of the bCA is a dimer where the active site is located at the interface of the two monomers (Kimber and Pai, 2000). In contrast, gCAs are trimers that have their active site zinc ion situated at the interface of two subunits coordinated by His residues from both subunits (Kisker et al, 1996;Iverson et al, 2000).In Arabidopsis (Arabidopsis thaliana), there are three gCA proteins and two g-like proteins that interact to form an extra structure of complex I of the mitochondrial electron transport chain (Perales et al, 2004;Sunderhaus et al, 2006). Although not active in vitro, gCA has been shown to bind inorganic carbon (Martin et al, 2009), affect complex I levels, plant growth, and gas-exchange rates when deleted (Perales et al, 2004;Soto et al, 2015), and cause plant sterility when ectopically overexpressed ).…”

“…The active unit of the bCA is a dimer where the active site is located at the interface of the two monomers (Kimber and Pai, 2000). In contrast, gCAs are trimers that have their active site zinc ion situated at the interface of two subunits coordinated by His residues from both subunits (Kisker et al, 1996;Iverson et al, 2000).…”

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

“…2D). The active site zinc of the ␣-and ␥-class enzymes is coordinated by three histidines and at least one water molecule (1,28,61). In the catalytic mechanism of carbonic anhydrase, the zinc-bound water ionizes to a metal-bound hydroxide ion that attacks CO 2 (equation 2a).…”

“…The ␥-class Cam is remarkably distinct from the ␣-class carbonic anhydrases in that it is a homotrimer in which each monomer adopts a novel left-handed ␤-helix fold (28,36). 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.…”