Crystallization, characterization and preliminary X-ray crystallographic analysis of GK2848, a putative carbonic anhydrase ofGeobacillus kaustophilus

Abstract

GK2848, a hypothetical protein from the thermophilic organism Geobacillus kaustophilus, was cloned and overexpressed in Escherichia coli. The protein was purified to homogeneity using Ni-NTA affinity-column and gel-filtration chromatography. The purified protein was crystallized using the sitting-drop vapour-diffusion method. The crystals diffracted to a resolution of 2.70 Å and belonged to the orthorhombic space group P2 1 2 1 2. GK2848 bears sequence homology to carbonic anhydrases of various bacterial species, indicating that it belongs to the carbonic anhydrase family of proteins. A subsequent carbonic anhydrase activity assay of GK2848 using the Wilbur-Anderson method confirmed its function as a carbonic anhydrase. A preliminary structure solution was obtained by molecular replacement using MOLREP. Mutation and biochemical characterization of the protein are in progress. The structure and functional analysis of GK2848 might provide valuable information on a novel class of carbonic anhydrases, as none of its homologous structures have been characterized.

Crystal Structure and Active Site Engineering of a Halophilic γ-Carbonic Anhydrase

“…To investigate the role of selected residues in the active site of CA_D, several variants were expressed, purified, and assayed for enzymatic activity. The selection of residues for mutagenesis was performed based on structural comparisons to γ-CA homologs as well as literature reports based on presumptions of conserved residues of γ-Cas (Smith et al, 1999;Iverson et al, 2000;Jeyakanthan et al, 2008;Ferry, 2010;Pena et al, 2010;Park et al, 2012;Ragunathan et al, 2013;Frost and McKenna, 2014). Thereby, the main comparison was focused on the Cam structure (Kisker et al, 1996).…”

Cloning and in silico analysis of a gene encoding a putative β-carbonic anhydrase from cowpea (Vigna unguiculata L. Walp)

“…Analysis of G-Factor of the modeled CuVA1 was noticed as −0.24, which revealed the quality of the predicted model (Table 3). Although there are several crystallographic structures of CA protein (Díaz Torres et al 2012;Ragunathan et al 2013); yet, we preferred to use P. sativum CA as a template for 3D VuCA1 protein modeling as it was one of the best characterized β-CA from C 3 dicotyledonous plant (Kimber & Pai 2000). The amino acid sequence alignment of two sequences showed high homology of 92.42%.…”