Centre of Advanced Study in Crystallography and Biophysics, University of Madras, Guindy Campus, Chennai, 600 025, India.
Department of Biological Sciences, Graduate School of Science, Osaka University, Toyonaka, Osaka, 560-0043, Japan.
Biochem Biophys Res Commun. 2021 Apr 2;547:96-101. doi: 10.1016/j.bbrc.2021.02.036. Epub 2021 Feb 17.
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 γ-class of CA (termed as Cag). Only a limited number of γ-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 γ-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 γ-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 CO to HCO when compared with Cam. Inductively Coupled Plasma - Optical Emission Spectrometry (ICP-OES) and Mg and Zn 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.
碳酸酐酶（CA）是已知最普遍的古老锌金属酶。在这里，我们报告了来自 Geobacillus kaustophilus 的假设蛋白 GK2848 的结构和功能分析。分析表明，它属于 CA 的γ类（称为 Cag）。迄今为止，只有少数 γ 类 CA 得到了表征。有趣的是，Cag 在其活性部位含有镁而不是传统的锌离子。基于与类似 γ-CA 的结构和序列比较，确定了 Cag 的假定活性位点残基。该分析表明，来自 Methanosarcina thermophila 的先前表征的γ-CA（Cam）的一个重要催化残基和质子穿梭残基（分别为 Glu62 和 Glu84）在 Cag 中缺失，但是 Cag 和 Cam 中都保守了某些其他活性位点残基。这表明与 Cam 相比，Cag 在 CO 到 HCO 的可逆水合过程中使用了不同的残基。对 Cag 及其突变体的电感耦合等离子体 - 光发射光谱（ICP-OES）和 Mg 和 Zn NMR 研究表明，Mg 或 Zn 都可以占据活性部位，这表明该酶具有交替性质。
