The CphAII protein from Aquifex aeolicus exhibits a metal-dependent phosphodiesterase activity.
The CphAII protein from the hyperthermophile Aquifex aeolicus shows the five conserved motifs of the metallo-β-lactamase (MBL) superfamily and presents 28% identity with the Aeromonas hydrophila subclass B2 CphA MBL. The gene encoding CphAII was amplified by PCR from the A. aeolicus genomic DNA and overexpressed in Escherichia coli using a pLex-based expression system. The recombinant CphAII protein was purified by a combination of heating (to denature E. coli proteins) and two steps of immobilized metal affinity chromatography. The purified enzyme preparation did not exhibit a β-lactamase activity but showed a metal-dependent phosphodiesterase activity versus bis-p-nitrophenyl phosphate and thymidine 5'-monophosphate p-nitrophenyl ester, with an optimum at 85°C. The circular dichroism spectrum was in agreement with the percentage of secondary structures characteristic of the MBL αββα fold.
Institute of Molecular Biotechnology, RWTH-Aachen University, Forckenbeckstraße 6, Aachen, Germany.
Michaël
M
Kupper
Cédric
C
Bauvois
Jean-Marie
JM
Frère
Kurt
K
Hoffmann
Moreno
M
Galleni
Carine
C
Bebrone
D000595:Amino Acid Sequence; D001419:Bacteria / Q000201:enzymology* / Q000235:genetics; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D002846:Chromatography, Affinity; D002942:Circular Dichroism; D017931:DNA Primers; D004269:DNA, Bacterial; D004591:Electrophoresis, Polyacrylamide Gel; D007700:Kinetics; D013058:Mass Spectrometry; D008670:Metals / Q000378:metabolism*; D008969:Molecular Sequence Data; D010727:Phosphoric Diester Hydrolases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid
D001426:Bacterial Proteins; D017931:DNA Primers; D004269:DNA, Bacterial; D008670:Metals; D011994:Recombinant Proteins; D010727:Phosphoric Diester Hydrolases