Structural and catalytic characterization of a thermally stable and acid-stable variant of human carbonic anhydrase II containing an engineered disulfide bond

Abstract

The carbonic anhydrases (CAs) are a family of mostly zinc metalloenzymes that catalyze the reversible hydration of CO2 to bicarbonate and a proton. Recently, there has been industrial interest in utilizing CAs as biocatalysts for carbon sequestration and biofuel production. The conditions used in these processes, however, result in high temperatures and acidic pH. This unfavorable environment results in rapid destabilization and loss of catalytic activity in CAs, ultimately resulting in cost-inefficient high-maintenance operation of the system. In order to negate these detrimental industrial conditions, cysteines at residues 23 (Ala23Cys) and 203 (Leu203Cys) were engineered into a wild-type variant of human CA II (HCAII) containing the mutation Cys206Ser. The X-ray crystallographic structure of the disulfide-containing HCAII (dsHCAII) was solved to 1.77 Å resolution and revealed that successful oxidation of the cysteine bond was achieved while also retaining desirable active-site geometry. Kinetic studies utilizing the measurement of (18)O-labeled CO2 by mass spectrometry revealed that dsHCAII retained high catalytic efficiency, and differential scanning calorimetry showed acid stability and thermal stability that was enhanced by up to 14 K compared with native HCAII. Together, these studies have shown that dsHCAII has properties that could be used in an industrial setting to help to lower costs and improve the overall reaction efficiency.

Structural, catalytic and stabilizing consequences of aromatic cluster variants in human carbonic anhydrase II

“…The proton-shuttle residue H64 (11) was seen to be in a dual conformation, ‘in’ and ‘out’, for the F226L and F226W variants, but was found only in the ‘in’ conformation (i.e., towards the active site) in the F226I variant. These variable conformations among crystallographic structures of HCA II are not an uncommon occurrence and are dependent on the crystallization conditions (1, 9, 10, 33, 34). …”

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

“…S5). The compact structure, the presence of an intramolecular disulfide bond and the dimeric arrangement of TcruCA are proposed to contribute significantly to the thermostability of the enzyme (Boone, Habibzadegan, Tu et al, 2013;Boone et al, 2015). Similar to other -CAs, the active site of TcruCA is subdivided into a hydrophilic and a hydrophobic pocket (Fig.…”

“…However, CA must maintain its characteristic high catalytic efficiency in extreme conditions, such as high temperature, pressure and extreme pHs, in order to be a viable CO 2 -sequestration agent (Savile & Lalonde, 2011;Boone, Habibzadegan, Gill et al, 2013). To this end, there has been a large effort to characterize CAs from organisms that thrive in extreme environments (Alvizo et al, 2014) and to engineer thermostable and pH-stable variants (Mirjafari et al, 2007;Boron, 2010;Yu et al, 2012;Boone, Habibzadegan, Tu et al, 2013). CAs are mainly zinc metalloenzymes that catalyze the interconversion of CO 2 and water to bicarbonate and a proton (Chegwidden et al, 2000;Duda & McKenna, 2006;Domsic et al, 2008).…”

“…These criteria alone would potentially make TcruCA, or any other dimeric -CA, a good candidate for industrial use owing to its attributed thermostability. To date, protein engineering of a thermostable hCA II variant has been focused on alterations in the monomeric structure of the enzyme (Boone, Habibzadegan, Gill et al, 2013;Boone, Habibzadegan, Tu et al, 2013;Boone et al, 2015). Although this has been successful in increasing the overall conformational stability of the enzyme, none of these variants have been utilized for industrial applications.…”

Acidic sweep gas with carbonic anhydrase coated hollow fiber membranes synergistically accelerates CO2 removal from blood

“…Bovine blood with Na-heparin anticoagulation (1:100 dilution of 1000U/mL) for gas exchange was purchased from Lampire Biological Laboratories (Pipersville, PA). Purified recombinant human carbonic anhydrase II was provided by Dr. Silverman and Dr. McKenna from University of Florida (Gainesville, Fl) [37]. Sulfur dioxide was purchased from Matheson Gas (Pittsburgh, PA).…”