Structural and biophysical characterization of the α-carbonic anhydrase from the gammaproteobacteriumThiomicrospira crunogenaXCL-2: insights into engineering thermostable enzymes for CO₂sequestration

Abstract

Biocatalytic CO 2 sequestration to reduce greenhouse-gas emissions from industrial processes is an active area of research. Carbonic anhydrases (CAs) are attractive enzymes for this process. However, the most active CAs display limited thermal and pH stability, making them less than ideal. As a result, there is an ongoing effort to engineer and/or find a thermostable CA to fulfill these needs. Here, the kinetic and thermal characterization is presented of an -CA recently discovered in the mesophilic hydrothermal vent-isolate extremophile Thiomicrospira crunogena XCL-2 (TcruCA), which has a significantly higher thermostability compared with human CA II (melting temperature of 71.9 C versus 59.5 C, respectively) but with a tenfold decrease in the catalytic efficiency. The X-ray crystallographic structure of the dimeric TcruCA shows that it has a highly conserved yet compact structure compared with other -CAs. In addition, TcruCA contains an intramolecular disulfide bond that stabilizes the enzyme. These features are thought to contribute significantly to the thermostability and pH stability of the enzyme and may be exploited to engineer -CAs for applications in industrial CO 2 sequestration.

The Structure of Carbonic Anhydrase IX Is Adapted for Low-pH Catalysis

“…In that regard, Diaz-Torres et al have proposed that CAs from extremophilic organisms have more compact structures that may underlie their enhanced stability. 31 However, the most striking or unusual characteristic of hCA IX-c is that it contains a surface far more net negatively charged than that of hCA II or XII (Table 4). This is revealed by observing the predicted theoretical pI of hCA IX-c [pI = 5.4 (Table 4)].…”

“… a Data from ref 31. b Data from ref 43. c Data from ref 7. Note that 18 O MS was not used to assay CA activity.…”

Activity and anion inhibition studies of the α-carbonic anhydrase from Thiomicrospira crunogena XCL-2 Gammaproteobacterium

“…4 Many of these bacterial CAs have displayed importance in terms of understanding microbial pathogenesis and metabolism, and also as a means to isolate thermo- and pH stable CAs that can be utilized as bio-catalytic agents for industrial CO 2 sequestration. 4,12,13 Such organisms include Vibrio cholera, 14 which is responsible for causing cholera in humans, and those found in hot springs or hydrothermal vents such as Sulfurihydrogenibium yellowstonense YO3AOP1, 15 Sulfurihydrogenibium azorense, 16 and Thiomicrospira crunogena XCL-2, 12 all observed to possess thermo- and pH stable CAs that can be utilized for industrial CO 2 sequestration.…”

“…Recently our group has determined the crystal structure of the α-CA from the gammaproteobacterium, Thiomicrospira crunogena XCL-2 (TcruCA; PDB ID: 4XZ5 12 ). Similar to other bacterial CAs, TcruCA has a high T M (72 °C) compared to human CA II (hCA II; 56 °C), poising its potential as a bio-catalyst for industrial CO 2 sequestration.…”

“…Similar to other bacterial CAs, TcruCA has a high T M (72 °C) compared to human CA II (hCA II; 56 °C), poising its potential as a bio-catalyst for industrial CO 2 sequestration. 12 The TcruCA monomer displays the signature secondary structure of an α-CA fold with helical and loop structures present toward the surface, and a conical active site cavity comprised of mostly β-strands surrounding a tetrahedrally coordinated (by three histidine residues) active site zinc ion (Figure 1). The overall characteristics of the active site of TcruCA exhibits defined hydrophobic and hydrophilic pockets similar to most other α-CAs (Figure 1).…”