Monofunctional domains of formiminotransferase-cyclodeaminase retain similar conformational stabilities outside the bifunctional octamer.
Each identical subunit of octameric formiminotransferase cyclodeaminase consists of a transferase and a deaminase domain connected by a short linker sequence. Both domains can be independently expressed in Escherichia coli as monofunctional dimers and show no indication of associating, suggesting that the linker mediates the only substantial interaction between the transferase and deaminase domains. To better understand the benefits arising from octamer formation, we have used equilibrium unfolding methods to examine the properties of the transferase and deaminase domains independently and within the octamer. Each isolated dimeric domain undergoes an apparent change in tertiary structure at low concentrations of urea (< 2 mol/l) which results in the concurrent loss of intrinsic fluorescence and catalytic activity. The full length octameric enzyme also undergoes inactivation and a loss of intrinsic fluorescence over this concentration range, without apparent change in secondary or quaternary structure. Between 2 and 2.5 M urea the isolated transferase and deaminase domains dissociate to monomers. However, only one of the subunit interfaces in the octamer is disrupted at this urea concentration and dissociation of the second interface occurs between 3.5 and 5 M urea. While each domain shows similar stability to denaturation within and outside of the octamer, one type of subunit interface achieves increased stability within the full length enzyme.
Murley
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LL
Department of Biochemistry, and Joint Centre for Structural Biology, Montreal, Que., Canada.
MacKenzie
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RE
D000642:Ammonia-Lyases / Q000648:ultrastructure*; D002942:Circular Dichroism; D004926:Escherichia coli; D046911:Macromolecular Substances; D011487:Protein Conformation; D011489:Protein Denaturation; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins; D013050:Spectrometry, Fluorescence; D014508:Urea
D046911:Macromolecular Substances; D011994:Recombinant Proteins; D014508:Urea; D000642:Ammonia-Lyases; C020148:formiminotetrahydrofolate cyclodeaminase