A plant-type (β-class) carbonic anhydrase in the thermophilic methanoarchaeon Methanobacterium thermoautotrophicum

Abstract

Carbonic anhydrase, a zinc enzyme catalyzing the interconversion of carbon dioxide and bicarbonate, is nearly ubiquitous in the tissues of highly evolved eukaryotes. Here we report on the first known plant-type (β-class) carbonic anhydrase in the archaea. The Methanobacterium thermoautotrophicum ΔH cab gene was hyperexpressed in Escherichia coli, and the heterologously produced protein was purified 13-fold to apparent homogeneity. The enzyme, designated Cab, is thermostable at temperatures up to 75°C. No esterase activity was detected with p-phenylacetate as the substrate. The enzyme is an apparent tetramer containing approximately one zinc per subunit, as determined by plasma emission spectroscopy. Cab has a CO2 hydration activity with a k(cat) of 1.7 x 104 s-1 and K(m) for CO2 of 2.9 mM at pH 8.5 and 25°C. Western blot analysis indicates that Cab (β class) is expressed in M. thermoautotrophicum; moreover, a protein cross-reacting to antiserum raised against the γ carbonic anhydrase from Methanosarcina thermophila was detected. These results show that β-class carbonic anhydrases extend not only into the Archaea domain but also into the thermophilic prokaryotes.