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Enzymes of Ancient Metabolic Pathways (dm)

We previously reported that carbonic anhydrases of the independently-evolved beta- and gamma-classes are widespread in procaryotes. We also reported that these enzymes have ancient origins, with the gamma-class dating to the origin of life. The novel nature of these discoveries prompted us to write the first review article on procaryotic carbonic anhydrases with the view to stimulate further research into these enzymes, which are likely to have played a role in the acquisition of carbon dioxide during the evolution of autotrophic pathways in early life forms. We also previously reported on the purification of a beta-class carbonic anhydrase from Methanobacterium thermoautotrophicum which is a thermophilic methane-producing archaeon. This current reporting period we continued our characterization of this enzyme, the first beta-class carbonic anhydrase from any procaryote. Kinetic analyses showed that the enzyme has a zinc-hydroxide mechanism common to the other two independently-evolved classes of carbonic anhydrases. The crystal structure was determined which revealed the overall fold and identified the active site. The structural results establish that the M. thermoautotrophicum enzyme represents a sub-class (termed the “cab-type”) of the beta-class, and that the beta-class is more diverse than either the alpha-class (predominantly found in mammals) or the gamma-class. The major difference between â??plant’ and â??cab’ type beta-class carbonic anhydrases is in the organization of the hydrophobic pocket thought to bind carbon dioxide. The active site architecture also identifies a route for transfer of a proton from the zinc-bound water molecule required for the zinc-hydroxide mechanism of catalysis. The overall results have provided further insight into the evolution of carbonic anhydrases and provided a foundation for continued studies to understand the catalytic mechanism and the roles they play in the autotrophic metabolism of deeply branching microbes in the phylogenetic tree of life.