Molecular structure of thermostable and zinc-ion-binding γ-class carbonic anhydrases
- 97 Downloads
The γ-class carbonic anhydrases (γ-CAs) mainly come from methanogens methane-producing bacteria that grow in hot springs and catalyze the interconversion of carbon dioxide and water to bicarbonate and protons. Here, the γ-CA from Thermus thermophilus HB8 (γ-TtCA) was expressed and purified, its crystal structure was determined at 2.3 Å resolution in space group P1. The asymmetric unit contains two trimers and six catalytic Zn2+. In general, the fold of the protein is similar to those of homologous enzymes from Geobacillus Kaustophilus, Bacillus Cereus, Methanosarcina Thermophila and others. Each monomer comprises a triangular prism-like structure consisting of a left-handed β-helix and a C-terminal α-helix. The catalytic Zn2+ bound to three histidines and a phosphate radical in a tetrahedral fashion. It is located at the interface between the two monomers. Inductively coupled plasma mass spectrometry measurements further suggest that the molar ratio of zinc ions and protein molecules is 1:1. The structure revealed a novel different region situated between the left-handed β-helix and the C-terminal α-helix. Compared to previously reported structures, half of the C-terminal α-helix was replaced with a long loop in this structure. The purified γ-TtCA exhibits no significant carbonic anhydrase activity compared to α-class carbonic anhydrases. This study provides insight into the structural diversity of γ-CAs with potential function for γ-CAs.
Keywordsγ-Class carbonic anhydrases Thermus thermophilus HB8 Crystal structure
The work was supported by the National Natural Science Foundation of China (Nos. 21601112 and 21671125), Talent Plan of Shanxi Province and Shanxi Scholarship Council of China (2015-021). We thank the staff at the BL17U1/18U1/19U1 beamline of National Center for Protein Sciences Shanghai (NCPSS) at Shanghai Synchrotron Radiation Facility for assistance during data collection. Crystallographic coordinates of γ-TtCAΔ169 are deposited to the Protein Data Bank with PDB code 6IVE.
Compliance with ethical standards
Conflict of interest
All of the authors of our manuscript declare that we have no competing interests.
- Adams PD, Afonine PV, Bunkoczi G, Chen VB, Davis IW, Echols N, Headd JJ, Hung LW, Kapral GJ, Grosse-Kunstleve RW, McCoy AJ, Moriarty NW, Oeffner R, Read RJ, Richardson DC, Richardson JS, Terwilliger TC, Zwart PH (2010) PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr Sect D 66:213–221. https://doi.org/10.1107/S0907444909052925 CrossRefGoogle Scholar
- Del PS, Vullo D, De LV, Carginale V, Scozzafava A, Supuran CT, Capasso C (2013) A highly catalytically active γ-carbonic anhydrase from the pathogenic anaerobe Porphyromonas gingivalis and its inhibition profile with anions and small molecules. Bioorg Med Chem Lett 23:4067–4071. https://doi.org/10.1016/j.bmcl.2013.05.063 CrossRefGoogle Scholar
- Delano WL (2009) The PyMOL molecular graphics system, version 188.8.131.52 Schrödinger, LLCGoogle Scholar
- Iino H, Naitow H, Nakamura Y, Nakagawa N, Agari Y, Kanagawa M, Ebihara A, Shinkai A, Sugahara M, Miyano M, Kamiya N, Yokoyama S, Hirotsu K, Kuramitsu S (2008) Crystallization screening test for the whole-cell project on Thermus thermophilus HB8. Acta Crystallogr Sect F 64(6):487–491. https://doi.org/10.1107/S1744309108013572 CrossRefGoogle Scholar
- Iverson TM, Alber BE, Kisker C, Ferry JG, Rees DC (2000) A closer look at the active site of gamma-class carbonic anhydrases: high-resolution crystallographic studies of the carbonic anhydrase from Methanosarcina thermophila. Biochemistry 39:9222–9231. https://doi.org/10.1021/bi000204s CrossRefPubMedGoogle Scholar
- Joseph P, Turtaut F, Ouahrani-Bettache S, Montero JL, Nishimori I, Minakuchi T, Vullo D, Scozzafava A, Kohler S, Winum JY, Supuran CT (2010) Cloning, characterization, and inhibition studies of a beta-carbonic anhydrase from Brucella suis. J Med Chem 53:2277–2285. https://doi.org/10.1021/jm901855h CrossRefPubMedGoogle Scholar
- Nishimori I, Minakuchi T, Morimoto K, Sano S, Onishi S, Takeuchi H, Vullo D, Scozzafava A, Supuran CT (2006) Carbonic anhydrase inhibitors: DNA cloning and inhibition studies of the alpha-carbonic anhydrase from Helicobacter pylori, a new target for developing sulfonamide and sulfamate gastric drugs. J Med Chem 49:2117–2126. https://doi.org/10.1021/jm0512600 CrossRefPubMedGoogle Scholar
- Nishimori I, Minakuchi T, Vullo D, Scozzafava A, Innocenti A, Supuran CT (2009) Carbonic anhydrase inhibitors. Cloning, characterization, and inhibition studies of a new beta-carbonic anhydrase from Mycobacterium tuberculosis. J Med Chem 52:3116–3120. https://doi.org/10.1021/jm9003126 CrossRefPubMedGoogle Scholar
- Rangarajan S, Jeyakanthan J, Mridula P, Kanaujia SP, Shiro Y, Kuramitsu S, Yokoyama S, Sekar K (2008) Observation of a calcium-binding site in the gamma-class carbonic anhydrase from Pyrococcus horikoshii. Acta Crystallogr Sect D 64:1012–1019. https://doi.org/10.1107/S0907444908024323 CrossRefGoogle Scholar
- Smith KS, Ferry JG (2000) Prokaryotic carbonic anhydrases. FEMS Microbiol Rev 24:335–366. https://doi.org/10.1111/j.1574-6976.2000.tb00546.x CrossRefPubMedGoogle Scholar