Substitutions of Thr-103-Ile and Trp-138-Gly in amidase from Pseudomonas aeruginosa are responsible for altered kinetic properties and enzyme instability.
Pseudomonas aeruginosa Ph1 is a mutant strain derived from strain AI3. The strain AI3 is able to use acetanilide as a carbon source through a mutation (T103I) in the amiE gene that encodes an aliphatic amidase (EC 3.5.1.4). The mutations in the amiE gene have been identified (Thr103Ile and Trp138Gly) by direct sequencing of PCR-amplified mutant gene from strain Ph1 and confirmed by sequencing the cloned PCR-amplified gene. Site-directed mutagenesis was used to alter the wild-type amidase gene at position 138 for Gly. The wild-type and mutant amidase genes (W138G, T103I-W138G, and T103I) were cloned into an expression vector and these enzymes were purified by affinity chromatography on epoxy-activated Sepharose 6B-acetamide/phenylacetamide followed by gel filtration chromatography. Altered amidases revealed several differences in kinetic properties, namely, in substrate specificity, sensitivity to urea, optimum pH, and enzyme stability, compared with the wild-type enzyme. The W138G enzyme acted on acetamide, acrylamide, phenylacetamide, and p-nitrophenylacetamide, whereas the double mutant (W138G and T103I) amidase acted only on p-nitrophenylacetamide and phenylacetamide. On the other hand, the T103I enzyme acted on p-nitroacetanilide and acetamide. The heat stability of altered enzymes revealed that they were less thermostable than the wild-type enzyme, as the mutant (W138G and W138G-T103I) enzymes exhibited t1/2 values of 7.0 and 1.5 min at 55 degrees C, respectively. The double substitution T103I and W138G on the amidase molecule was responsible for increased instability due to a conformational change in the enzyme molecule as detected by monoclonal antibodies. This conformational change in altered amidase did not alter its M(r) value and monoclonal antibodies reacted differently with the active and inactive T103I-W138G amidase.
Karmali
A
A
Laboratório de Engenharia Bioquímica do Departamento de Engenharia Química do Instituto Superior de Engenharia de Lisboa, Rua Conselheiro Emídio Navarro, 1900 Lisboa-Portugal.
Pacheco
R
R
Tata
R
R
Brown
P
P
D000581:Amidohydrolases / Q000737:chemistry* / Q000235:genetics*; D004797:Enzyme-Linked Immunosorbent Assay; D005998:Glycine / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D007532:Isoleucine / Q000737:chemistry; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010957:Plasmids; D016133:Polymerase Chain Reaction; D011550:Pseudomonas aeruginosa / Q000201:enzymology* / Q000235:genetics*; D013696:Temperature; D013912:Threonine / Q000737:chemistry; D014364:Tryptophan / Q000737:chemistry; D014508:Urea / Q000494:pharmacology
D007532:Isoleucine; D013912:Threonine; D014364:Tryptophan; D014508:Urea; D000581:Amidohydrolases; C019665:amidase; D005998:Glycine