Reconstitution of carbonic anhydrase activity of the cell-surface-binding protein of vaccinia virus.
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.
Ohradanova
Anna
A
Centre of Molecular Medicine, Institute of Virology, Slovak Academy of Sciences, Dubravska cesta 9, 845 05 Bratislava, Slovak Republic.
Vullo
Daniela
D
Kopacek
Juraj
J
Temperini
Claudia
C
Betakova
Tatiana
T
Pastorekova
Silvia
S
Pastorek
Jaromir
J
Supuran
Claudiu T
CT
D000086:Acetazolamide / Q000494:pharmacology; D000595:Amino Acid Sequence; D001665:Binding Sites; D002256:Carbonic Anhydrases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D006367:HeLa Cells; D006639:Histidine / Q000235:genetics / Q000378:metabolism; D006801:Humans; D008969:Molecular Sequence Data; D009154:Mutation; D010802:Phylogeny; D015202:Protein Engineering; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D016415:Sequence Alignment; D014162:Transfection; D014616:Vaccinia virus / Q000201:enzymology* / Q000378:metabolism; D014764:Viral Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*
D011994:Recombinant Proteins; D014764:Viral Proteins; D006639:Histidine; D002256:Carbonic Anhydrases; D000086:Acetazolamide