Substitution of Glu-59 by Val in amidase from Pseudomonas aeruginosa results in a catalytically inactive enzyme.
A mutant strain, KLAM59, of Pseudomonas aeruginosa has been isolated that synthesizes a catalytically inactive amidase. The mutation in the amidase gene has been identified (Glu59Val) by direct sequencing of PCR-amplified mutant gene and confirmed by sequencing the cloned PCR-amplified gene. The wild-type and altered amidase genes were cloned into an expression vector and both enzymes were purified by affinity chromatography on epoxy-activated Sepharose 6B-acetamide followed by gel filtration chromatography. The mutant enzyme was catalytically inactive, and it was detected in column fractions by monoclonal antibodies previously raised against the wild-type enzyme using an ELISA sandwich method. The recombinant wild-type and mutant enzymes were purified with a final recovery of enzyme in the range of 70-80%. The wild-type and mutant enzymes behaved differently on the affinity column as shown by their elution profiles. The molecular weights of the purified wild-type and mutant amidases were found to be 210,000 and 78,000 Dalton, respectively, by gel filtration chromatography. On the other hand, the mutant enzyme ran as a single protein band on SDS-PAGE and native PAGE with a M(r) of 38,000 and 78,000 Dalton, respectively. These data suggest that the substitution Glu59Val was responsible for the dimeric structure of the mutant enzyme as opposed to the hexameric form of the wild-type enzyme. Therefore, the Glu59 seems to be a critical residue in the maintenance of the native quaternary structure of amidase.
Karmali
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Departamento de Engenharia Química do Instituto Superior de Engenharia de Lisboa, Portugal. akarmali@scientist.com
Tata
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Brown
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D000581:Amidohydrolases / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D019943:Amino Acid Substitution; D002384:Catalysis; D002846:Chromatography, Affinity / Q000379:methods; D002850:Chromatography, Gel / Q000379:methods; D003001:Cloning, Molecular / Q000379:methods; D017931:DNA Primers; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D004926:Escherichia coli / Q000235:genetics; D005784:Gene Amplification; D018698:Glutamic Acid; D007700:Kinetics; D008970:Molecular Weight; D016133:Polymerase Chain Reaction / Q000379:methods; D011550:Pseudomonas aeruginosa / Q000201:enzymology*; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D014633:Valine
D017931:DNA Primers; D011994:Recombinant Proteins; D018698:Glutamic Acid; D000581:Amidohydrolases; C019665:amidase; D014633:Valine