Structural and functional characterization of a putative carbonic anhydrase from Geobacillus kaustophilus reveals its cambialistic function

Carbonic anhydrases (CA) are the most ubiquitous ancient zinc metalloenzymes known. Here we report the structural and functional analysis of a hypothetical protein GK2848 from Geobacillus kaustophilus. The analysis revealed that it belongs to the gamma-class of CA (termed as Cag). Only a limited number of gamma-class CA's have been characterized till date. Interestingly Cag contains magnesium at its active site instead of a traditional zinc ion. Based on the structural and sequence comparison with similar gamma-CA's the putative active site residues of Cag were identified. This analysis revealed that an important catalytic residue and a proton shuttle residue (Glu62 and Glu84 respectively) of Cam (previously characterized gamma-CA from Methanosarcina thermophila) are absent in Cag, however certain other active site residues are conserved both in Cag and Cam. This suggests that Cag uses a different set of residues for the reversible hydration of CO2 to HCO3- when compared with Cam. Inductively Coupled Plasma - Optical Emission Spectrometry (ICP-OES) and Mg-25 and Zn-67 NMR studies on Cag and its mutants revealed that either Mg or Zn can occupy the active site which suggests the cambialistic nature of the enzyme. (c) 2021 Elsevier Inc. All rights reserved.

Automated summary

Carbonic anhydrases (CA) are a group of zinc metalloenzymes, with a newly identified gamma-class CA (Cag) from Geobacillus kaustophilus containing magnesium at its active site instead of the traditional zinc ion. While lacking some essential catalytic residues present in other gamma-CA's, Cag still shares certain conserved active site residues with characterized enzymes. Inductive Coupled Plasma - Optical Emission Spectrometry (ICP-OES) and NMR studies revealed the cambialistic nature of Cag, with the ability to bind both magnesium and zinc at its active site.