The N-terminal region of a 32 kDa cell-surface-binding protein, encoded by the D8L gene of vaccinia virus, shows sequence homology to CAs (carbonic anhydrases; EC 4.2.1.1). The active CAs catalyse the reversible hydration of CO2 to bicarbonate participating in many physiological processes. The CA-like domain of vaccinia protein [vaccCA (vaccinia virus CA-like protein)] contains one of the three conserved histidine residues required for co-ordination to the catalytic zinc ion and for enzyme activity. In the present study, we report the engineering of catalytically active vaccCA mutants by introduction of the missing histidine residues into the wild-type protein. The wild-type vaccCA was inactive as a catalyst and does not bind sulfonamide CA inhibitors. Its position on a phylogram with other hCAs (human CAs) shows a relationship with the acatalytic isoforms CA X and XI, suggesting that the corresponding viral gene was acquired from the human genome by horizontal gene transfer. The single mutants (vaccCA N92H/Y69H) showed low enzyme activity and low affinity for acetazolamide, a classical sulfonamide CA inhibitor. The activity of the double mutant, vaccCA N92H/Y69H, was much higher, of the same order of magnitude as that of some human isoforms, namely CA VA and CA XII. Moreover, its affinity for acetazolamide was high, comparable with that of the most efficient human isoenzyme, CA II (in the low nanomolar range). Multiplication of vaccinia virus in HeLa cells transfected with the vaccCA N92H/Y69H double mutant was approx. 2-fold more efficient than in wild-type vaccCA transfectants, suggesting that the reconstitution of the enzyme activity improved the virus life cycle.
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September 12 2007
Reconstitution of carbonic anhydrase activity of the cell-surface-binding protein of vaccinia virus Available to Purchase
Anna Ohradanova;
Anna Ohradanova
*Centre of Molecular Medicine, Institute of Virology, Slovak Academy of Sciences, Dubravska cesta 9, 845 05 Bratislava, Slovak Republic
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Daniela Vullo;
Daniela Vullo
†Università degli Studi di Firenze, Polo Scientifico, Laboratorio di Chimica Bioinorganica, Rm. 188, Via della Lastruccia 3, 50019 Sesto Fiorentino, Italy
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Juraj Kopacek;
Juraj Kopacek
*Centre of Molecular Medicine, Institute of Virology, Slovak Academy of Sciences, Dubravska cesta 9, 845 05 Bratislava, Slovak Republic
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Claudia Temperini;
Claudia Temperini
†Università degli Studi di Firenze, Polo Scientifico, Laboratorio di Chimica Bioinorganica, Rm. 188, Via della Lastruccia 3, 50019 Sesto Fiorentino, Italy
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Tatiana Betakova;
Tatiana Betakova
*Centre of Molecular Medicine, Institute of Virology, Slovak Academy of Sciences, Dubravska cesta 9, 845 05 Bratislava, Slovak Republic
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Silvia Pastorekova;
Silvia Pastorekova
*Centre of Molecular Medicine, Institute of Virology, Slovak Academy of Sciences, Dubravska cesta 9, 845 05 Bratislava, Slovak Republic
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Jaromir Pastorek;
Jaromir Pastorek
1
*Centre of Molecular Medicine, Institute of Virology, Slovak Academy of Sciences, Dubravska cesta 9, 845 05 Bratislava, Slovak Republic
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Claudiu T. Supuran
†Università degli Studi di Firenze, Polo Scientifico, Laboratorio di Chimica Bioinorganica, Rm. 188, Via della Lastruccia 3, 50019 Sesto Fiorentino, Italy
2To whom correspondence should be addressed (email [email protected]).
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Publisher: Portland Press Ltd
Received:
June 20 2007
Accepted:
July 06 2007
Accepted Manuscript online:
July 06 2007
Online ISSN: 1470-8728
Print ISSN: 0264-6021
© The Authors Journal compilation © 2007 Biochemical Society
2007
Biochem J (2007) 407 (1): 61–67.
Article history
Received:
June 20 2007
Accepted:
July 06 2007
Accepted Manuscript online:
July 06 2007
Citation
Anna Ohradanova, Daniela Vullo, Juraj Kopacek, Claudia Temperini, Tatiana Betakova, Silvia Pastorekova, Jaromir Pastorek, Claudiu T. Supuran; Reconstitution of carbonic anhydrase activity of the cell-surface-binding protein of vaccinia virus. Biochem J 1 October 2007; 407 (1): 61–67. doi: https://doi.org/10.1042/BJ20070816
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