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

The X-ray crystallographic structure of the disulfide-containing HCAII (dsHCAII) has been solved to 1.77 Å resolution and revealed that successful oxidation of the cysteine bond was achieved while also retaining desirable active-site geometry. The carbonic anhydrases (CAs) are a family of mostly zinc metalloenzymes that catalyze the reversible hydration of CO{sub 2} 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 {sup 18}O-labeled CO{sub 2} by mass spectrometry revealed that dsHCAII retained high catalytic efficiency, and differential scanning calorimetry showed acidmore » 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.« less
Authors:
;  [1] ; ;  [2] ;  [1]
  1. University of Florida, PO Box 100245, Gainesville, FL 32610 (United States)
  2. University of Florida, PO Box 100267, Gainesville, FL 32610 (United States)
Publication Date:
OSTI Identifier:
22351291
Resource Type:
Journal Article
Resource Relation:
Journal Name: Acta Crystallographica. Section D: Biological Crystallography; Journal Volume: 69; Journal Issue: Pt 8; Other Information: PMCID: PMC3727326; PMID: 23897465; PUBLISHER-ID: be5229; OAI: oai:pubmedcentral.nih.gov:3727326; Copyright (c) International Union of Crystallography 2013; Country of input: International Atomic Energy Agency (IAEA)
Country of Publication:
Denmark
Language:
English
Subject:
75 CONDENSED MATTER PHYSICS, SUPERCONDUCTIVITY AND SUPERFLUIDITY; CALORIMETRY; CYSTEINE; EFFICIENCY; ENVIRONMENT; GEOMETRY; HYDRATION; IRON; MASS SPECTROSCOPY; OXIDATION; PROTONS; RESOLUTION; STABILITY; TEMPERATURE RANGE 0400-1000 K; ZINC