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Crystal Structure of a Highly Thermostable ?-Carbonic Anhydrase from Persephonella marina EX-H1.

ABSTRACT: Bacterial ?-type carbonic anhydrase (?-CA) is a zinc metalloenzyme that catalyzes the reversible and extremely rapid interconversion of carbon dioxide to bicarbonate. In this study, we report the first crystal structure of a hyperthermostable ?-CA from Persephonella marina EXH1 (pm CA) in the absence and presence of competitive inhibitor, acetazolamide. The structure reveals a compactly folded pm CA homodimer in which each monomer consists of a 10-stranded ?-sheet in the center. The catalytic zinc ion is coordinated by three highly conserved histidine residues with an exchangeable fourth ligand (a water molecule, a bicarbonate anion, or the sulfonamide group of acetazolamide). Together with an intramolecular disulfide bond, extensive interfacial networks of hydrogen bonds, ionic and hydrophobic interactions stabilize the dimeric structure and are likely responsible for the high thermal stability. We also identified novel binding sites for calcium ions at the crystallographic interface, which serve as molecular glue linking negatively charged and otherwise repulsive surfaces. Furthermore, this large negatively charged patch appears to further increase the thermostability at alkaline pH range via favorable charge-charge interactions between pm CA and solvent molecules. These findings may assist development of novel ?-CAs with improved thermal and/or alkaline stability for applications such as CO2 capture and sequestration.

SUBMITTER: Kim S

PROVIDER: S-EPMC6602146 | biostudies-literature | 2019 Jun

REPOSITORIES: biostudies-literature

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Crystal Structure of a Highly Thermostable α-Carbonic Anhydrase from Persephonella marina EX-H1.

Kim Subin S Sung Jongmin J Yeon Jungyoon J Choi Seung Hun SH Jin Mi Sun MS

Molecules and cells 20190601 6

Bacterial α-type carbonic anhydrase (α-CA) is a zinc metalloenzyme that catalyzes the reversible and extremely rapid interconversion of carbon dioxide to bicarbonate. In this study, we report the first crystal structure of a hyperthermostable α-CA from Persephonella marina EXH1 (<i>pm</i> CA) in the absence and presence of competitive inhibitor, acetazolamide. The structure reveals a compactly folded pm CA homodimer in which each monomer consists of a 10-stranded β-sheet in the center. The catal ...[more]

PMID: 31250619

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Project description:BACKGROUND:The pathogenic yeast Candida albicans can proliferate in environments with different carbon dioxide concentrations thanks to the carbonic anhydrase CaNce103p, which accelerates spontaneous conversion of carbon dioxide to bicarbonate and vice versa. Without functional CaNce103p, C. albicans cannot survive in atmospheric air. CaNce103p falls into the ?-carbonic anhydrase class, along with its ortholog ScNce103p from Saccharomyces cerevisiae. The crystal structure of CaNce103p is of interest because this enzyme is a potential target for surface disinfectants. RESULTS:Recombinant CaNce103p was prepared in E. coli, and its crystal structure was determined at 2.2 Å resolution. CaNce103p forms a homotetramer organized as a dimer of dimers, in which the dimerization and tetramerization surfaces are perpendicular. Although the physiological role of CaNce103p is similar to that of ScNce103p from baker's yeast, on the structural level it more closely resembles carbonic anhydrase from the saprophytic fungus Sordaria macrospora, which is also tetrameric. Dimerization is mediated by two helices in the N-terminal domain of the subunits. The N-terminus of CaNce103p is flexible, and crystals were obtained only upon truncation of the first 29 amino acids. Analysis of CaNce103p variants truncated by 29, 48 and 61 amino acids showed that residues 30-48 are essential for dimerization. Each subunit contains a zinc atom in the active site and displays features characteristic of type I ?-carbonic anhydrases. Zinc is tetrahedrally coordinated by one histidine residue, two cysteine residues and a molecule of ?-mercaptoethanol originating from the crystallization buffer. The active sites are accessible via substrate tunnels, which are slightly longer and narrower than those observed in other fungal carbonic anhydrases. CONCLUSIONS:CaNce103p is a ?-class homotetrameric metalloenzyme composed of two homodimers. Its structure closely resembles those of other ?-type carbonic anhydrases, in particular CAS1 from Sordaria macrospora. The main differences occur in the N-terminal part and the substrate tunnel. Detailed knowledge of the CaNce103p structure and the properties of the substrate tunnel in particular will facilitate design of selective inhibitors of this enzyme.

Dataset Information

Crystal Structure of a Highly Thermostable ?-Carbonic Anhydrase from Persephonella marina EX-H1.

Publications

Crystal Structure of a Highly Thermostable α-Carbonic Anhydrase from Persephonella marina EX-H1.

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