The interaction of Fusarium solani pisi cutinase with long chain spin label esters.
We here present a study of the interaction between the Fusarium solani pisi cutinase mutant S120A and spin-labeled 4,4-dimethyloxazoline-N-oxyl-(DOXYL)-stearoyl-glycerol substrates in a micellar system. The interaction is detected by NMR measuring changes in chemical shift for 1H and 15N as well as relaxation parameters for backbone 1H (T1) and 15N (T1, T2) atoms as well as for side chain methyl groups 1H (T1). The detected interaction shows a weak binding of cutinase to the lipid micelles. Structural and mobility changes are located inside and around the active site, its flanking loops, and the oxyanion hole, respectively. Relaxation changes in the amino acid pairs Ser 92, Ala 93 and Thr 173, Gly 174 positioned at the edge of each of the active site flanking loops make these residues prime candidates for hinges, allowing for structural rearrangement during substrate binding. The cutinase mutant S120A used carries a 15 amino acid pro-peptide; the significance of this pro-peptide was so far undetermined. We show here that the pro-peptide is affected by the presence of the micellar substrate. Relaxation enhancements indicative of spatial proximity between the DOXYL group in the lipid chain and some hydrophobic residues surrounding the active site could be found.
Poulsen
Kristian R
KR
Biostructure and Protein Engineering Group, Department of Physics and Nanotechnology, Aalborg University, DK-9220 Aalborg, Denmark.
Sørensen
Thomas K
TK
Duroux
Laurent
L
Petersen
Evamaria I
EI
Petersen
Steffen B
SB
Wimmer
Reinhard
R
D000409:Alanine / Q000235:genetics; D002265:Carboxylic Ester Hydrolases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D003497:Cyclic N-Oxides / Q000737:chemistry; D004578:Electron Spin Resonance Spectroscopy; D004952:Esters; D005609:Free Radicals / Q000378:metabolism; D005656:Fungal Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D005670:Fusarium / Q000201:enzymology* / Q000235:genetics; D008823:Micelles; D019906:Nuclear Magnetic Resonance, Biomolecular; D017510:Protein Folding; D017433:Protein Structure, Secondary; D012694:Serine / Q000235:genetics; D013113:Spin Labels; D013379:Substrate Specificity
D003497:Cyclic N-Oxides; D004952:Esters; D005609:Free Radicals; D005656:Fungal Proteins; D008823:Micelles; D013113:Spin Labels; C005072:5-doxylstearic acid; D012694:Serine; C032363:16-nitroxystearic acid; D002265:Carboxylic Ester Hydrolases; C019952:cutinase; D000409:Alanine