Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.5.1.4 (deaminase)
 5,113 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Hydroxyurea inhibited growth of Pseudomonas aeruginosa strain AI 3 on media containing either acetanilide (N-phenyl acetamide) or acetamide as sole carbon sources. Mutants resistant to hydroxyurea inhibition of growth on acetanilide (OUCH strains) and acetamide (AmOUCH strains) displayed altered growth properties on various amide media compared with the parent strain AI3. AI3 amidase, which catalyses the initial step in the metabolism of acetanilide and acetamide, was inhibited by hydroxyurea in a time-dependent reaction that was slowly reversible at pH 7.2. Compared with AI3 amidase, amidases from the OUCH mutants were much less sensitive to inhibition by hydroxyurea and showed altered substrate specificities and pH/activity profiles; amidases from the AmOUCH mutants were more sensitive to hydroxyurea inhibition but showed increased activity towards acetamide. Association of resistance to hydroxyurea inhibition with a mutation in the amidase structural gene of strain OUCH 4 was confirmed by transduction.
 ...
PMID:Relationship between mutant amidases of Pseudomonas aeruginosa and hydroxyurea as an inhibitor. 10 40

Amidases (acylamide amidohydrolase EC 3.5.1.4) from mutant strains (i.e., B6, AI3, AIU1N, OUCH 4 and L10) of Pseudomonas aeruginosa were purified in one-step by ligand affinity chromatography using Epoxy-activated Sepharose 4B-acetamide. The yields of the purified enzymes were about 90% for all mutant strains with purification factors of about 10 and were apparently homogeneous when analysed by SDS-PAGE and native PAGE. The protein bands on native PAGE coincided with the stained band of enzyme activity for all amidase preparations. Affinity columns had a maximum binding capacity of 0.5 mg amidase protein/ml of sedimented gel and could be regenerated and reused several times without any loss of binding capacity and resolution. Affinity gels containing either semicarbazide or urea were also found useful for the isolation of amidase. The differences in substrate specificity of these amidases reported previously were also observed in the elution behaviour of these enzymes from the affinity columns.
 ...
PMID:One-step affinity purification of amidase from mutant strains of Pseudomonas aeruginosa. 251 78

Mutants able to utilize phenylacetamide as sole nitrogen source were isolated from the acetanilide (N-phenylacetamide) - utilizing Pseudomonas aeruginosa mutant strain A13 and from its parent strain L 10. Growth properties of the mutants (Ph strains) on amide media and the physicochemical properties of their amidases in cell free extracts indicated that their phenylacetamidase activities were attributable to alterations in their amidases. Differences in amide hydrolase specificities between the AI3- and the L 10-Ph mutants were observed. The AI3 group had a high level of activity towards 4-nitrophenylacetamide, activity towards phenylacetyl-4-nitroaniline but, unlike strain AI3, no activity towards acetyl-4-nitroaniline; the L 10 group had a low activity towards 4-nitrophenylacetamide, no activity towards phenylacetyl-4-nitroaniline but retained the low level of activity towards acetyl-4-nitroaniline exhibited by strain L 10. Confirmation of the association between these altered specificities and alterations in amidases was obtained from analysis of the properties of phenylacetamidases purified from an AI3-Ph mutant (pH 5) and an L 10-Ph mutant (Ph14). The original mutation in the amidase gene of strain AI3 appeared responsible for the differences between the two groups of Ph mutants and the binding interactions with acetanilide that it determined were eliminated in AI3-Ph mutants.
 ...
PMID:Adaptation to phenylacetamide as a growth substrate by an acetanilide-utilizing mutant of Pseudomonas aeruginosa. 676 19

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.
 ...
PMID:Substitutions of Thr-103-Ile and Trp-138-Gly in amidase from Pseudomonas aeruginosa are responsible for altered kinetic properties and enzyme instability. 1143 8