A failed tentative to design a super carbonic anhydrase having the biochemical properties of the most thermostable CA (SspCA) and the fastest (SazCA) enzymes

Carbonic anhydrases (CAs, EC 4.2.1.1) catalyze a simple reaction in all life domains: the carbon dioxide hydration to bicarbonate and protons: CO2 + H2O -> HCO + H+. Six different, genetically distinct CA families are known to date, the alpha-, beta-, gamma-, delta-, zeta- and Z-CAs. Bacteria encode for CAs belong to the alpha-, beta- and gamma-classes. Recently, our groups investigated the presence of CAs in two bacteria belonging to the genus Sulfurihydrogenibium living in hot springs all over the world, at temperatures of up to 110 degrees C. The alpha-CAs from Sulfurihydrogenibium yellow-stonense and Sulfurihydrogenibium azorense, denominated SspCA and SazCA, respectively, are highly thermostable, maintaining a good catalytic activity even after being heated for a prolonged period. Moreover, SazCA was to be the fastest CA known to date with a k(cat) value of 4.40 x 10(6) s(-1) and a k(cat)/K-M value of 3.5 x 10(8) M-1 s(-1). SspCA also showed a good catalytic activity for the same reaction, with a k(cat) value of 9.35 x 10(5) s(-1) and a k(cat)/K-M value of 1.1 x 10(8) M-1 s(-1), proving that the extremo-alpha-CAs'' are between the most effective CAs known to date. Here, we describe a failed tentative to obtain a super-CA, SupCA, by combining the amino acid sequence of SazCA and SspCA. To achieve this goal we introduced six His residues in N-terminal sequence of SspCA. However the obtained SupCA showed lower catalytic activity and thermostability compared to both extremophilic enzymes from which it has been designed. We rationalized the biochemical reasons of this failure, which may be useful to design enzymes with a better catalytic activity.