Use of a designed fusion protein dissociates allosteric properties from the dodecameric state of Pseudomonas aeruginosa catabolic ornithine carbamoyltransferase.
The catabolic ornithine carbamoyltransferase from Pseudomonas aeruginosa, an enzyme consisting of 12 identical 38-kDa subunits, displays allosteric properties, namely carbamoylphosphate homotropic cooperativity and heterotropic activation by AMP and other nucleoside monophosphates and inhibition by polyamines. To shed light on the effect of the oligomeric organization on the enzyme's activity and/or allosteric behavior, a hybrid ornithine carbamoyltransferase/glutathione S-transferase (OTCase-GST) molecule was constructed by fusing the 3' end of the P. aeruginosa arcB gene (OTCase) to the 5' end of the cDNA encoding Musca domestica GST by using a polyglycine encoding sequence as a linker. The fusion protein was overexpressed in Escherichia coli and purified from cell extracts by affinity chromatography, making use of the GST domain. It was found to exist as a trimer and to retain both the homotropic and heterotropic characteristic interactions of the wild-type catabolic OTCase but to a lower extent as compared with the wild-type OTCase. The dodecameric organization of catabolic P. aeruginosa OTCase may therefore be related to an enhancement of the substrate cooperativity already present in its trimers (and perhaps also to the thermostability of the enzyme).
Mouz
N
N
Laboratoire de Cristallographie Macromoléculaire, Institut de Biologie Structurale Jean-Pierre EBEL, Commissariat à l'Energie Atomique-Centre National de la Recherche Scientifique, Grenoble, France.
Tricot
C
C
Ebel
C
C
Petillot
Y
Y
Stalon
V
V
Dideberg
O
O
D000494:Allosteric Regulation; D001483:Base Sequence; D015195:Drug Design; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability; D005982:Glutathione Transferase; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009954:Ornithine Carbamoyltransferase / Q000737:chemistry / Q000235:genetics*; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D011550:Pseudomonas aeruginosa / Q000201:enzymology*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D015183:Restriction Mapping
D011993:Recombinant Fusion Proteins; D009954:Ornithine Carbamoyltransferase; D005982:Glutathione Transferase