1H, 13C, and 15N resonance assignments of Fusarium solani pisi cutinase and preliminary features of the structure in solution.
Essentially complete (96%) sequence-specific assignments were made for the backbone and side-chain 1H, 13C, and 15N resonances of Fusarium solani pisi cutinase, produced as a 214-residue heterologous protein in Escherichia coli, using heteronuclear NMR techniques. Three structural features were noticed during the assignment. (1) The secondary structure in solution corresponds mostly with the structure from X-ray diffraction, suggesting that both structures are globally similar. (2) The HN of Ala32 has a strongly upfield-shifted resonance at 3.97 ppm, indicative of an amide-aromatic hydrogen bond to the indole ring of Trp69 that stabilizes the N-terminal side of the parallel beta-sheet. (3) The NMR data suggest that the residues constituting the oxyanion hole are quite mobile in the free enzyme in solution, in contrast to the existence of a preformed oxyanion hole as observed in the crystal structure. Apparently, cutinase forms its oxyanion hole upon binding of the substrate like true lipases.
Prompers
J J
JJ
Nijmegen SON Research Center, Laboratory of Biophysical Chemistry, University of Nijmegen, The Netherlands.
Groenewegen
A
A
Van Schaik
R C
RC
Pepermans
H A
HA
Hilbers
C W
CW
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000737:chemistry; D002247:Carbon Isotopes; D002265:Carboxylic Ester Hydrolases / Q000737:chemistry*; D005670:Fusarium / Q000201:enzymology*; D006859:Hydrogen; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009587:Nitrogen Isotopes; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods; D017433:Protein Structure, Secondary; D012996:Solutions
D000596:Amino Acids; D002247:Carbon Isotopes; D009587:Nitrogen Isotopes; D012996:Solutions; D006859:Hydrogen; D002265:Carboxylic Ester Hydrolases; C019952:cutinase