Biochemical and structural characterisation of cutinase mutants in the presence of the anionic surfactant AOT.
The reactivity, stability and unfolding of wild-type (WT) Fusarium solani pisi cutinase and L153Q, S54D and T179C variants were studied in the absence and presence of the dioctyl sulfosuccinate sodium salt (AOT) surfactant. In the absence of surfactant the S54D variant catalytic activity is similar to that of the WT cutinase, whereas L153Q and T179C variants show a lower activity. AOT addition induces an activity reduction for WT cutinase and its variants, although for low AOT concentrations a small increase of activity was observed for S54D and T179C. The enzyme deactivation in the presence of 0.5 mM AOT is relatively slow for the S54D and T179C variants when compared to wild-type cutinase and L153Q variant. These results were correlated with secondary and tertiary structure changes assessed by the CD spectrum and fluorescence of the single tryptophan and the six tyrosine residues. The WT cutinase and S54D variant have similar secondary and tertiary structures that differ from those of T179C and L153Q variants. L153Q, S54D and T179C mutations prevent the formation of hydrophobic crevices responsible for the unfolding by anionic surfactants, with the consequent decrease of the AOT-cutinase interactions.
Brissos
V
V
IBB - Institute for Biotechnology and Bioengineering, Centre for Biological and Chemical Engineering, Instituto Superior Técnico, Av. Rovisco Pais, 1049-001 Lisboa, Portugal. vaniabrissos@ist.utl.pt
Melo
E P
EP
Martinho
J M G
JM
Cabral
J M S
JM
D019943:Amino Acid Substitution; D000817:Anilino Naphthalenesulfonates / Q000378:metabolism; D002087:Butyrates / Q000378:metabolism; D002265:Carboxylic Ester Hydrolases / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D002942:Circular Dichroism; D004795:Enzyme Stability / Q000187:drug effects; D005656:Fungal Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D005670:Fusarium / Q000201:enzymology / Q000235:genetics; D006868:Hydrolysis; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D013386:Succinates / Q000494:pharmacology; D013501:Surface-Active Agents / Q000494:pharmacology; D013816:Thermodynamics
C512554:1,4-bis(2-ethylhexyl) sodium sulfosuccinate; D000817:Anilino Naphthalenesulfonates; D002087:Butyrates; D005656:Fungal Proteins; D011994:Recombinant Proteins; D013386:Succinates; D013501:Surface-Active Agents; C033592:4-nitrophenyl butyrate; C027132:1-anilino-8-naphthalenesulfonate; D002265:Carboxylic Ester Hydrolases; C019952:cutinase