Open AccessCrystallization, characterization and preliminary X‐ray crystallographic analysis of GK2848, a putative carbonic anhydrase of Geobacillus kaustophilus
Author(s) -
Ragunathan Preethi,
Raghunath Gokul,
Kuramitsu Seiki,
Yokoyama Shigeyuki,
Kumarevel Thirumananseri,
Ponnuraj Karthe
Publication year - 2013
Publication title -
acta crystallographica section f
Language(s) - English
Resource type - Journals
ISSN - 1744-3091
DOI - 10.1107/s1744309112051913
Subject(s) - carbonic anhydrase , biochemistry , escherichia coli , chemistry , carbonic anhydrase ii , size exclusion chromatography , thermophile , orthorhombic crystal system , biology , enzyme , crystallography , crystal structure , gene
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 P 2 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.