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Use of a designed fusion protein dissociates allosteric properties from the dodecameric state of Pseudomonas aeruginosa catabolic ornithine carbamoyltransferase

par Mouz, Nicolas;Tricot, Catherine ;Ebel, Christine;Petillot, Yves;Stalon, Victor ;Dideberg, O.
Référence Proceedings of the National Academy of Sciences of the United States of America, 93, 18, page (9414-9419)
Publication Publié, 1996-09

Article révisé par les pairs

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Résumé :

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 mono-phosphates 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 carbamoyl-transferase/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 OT-Case 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).