¹H, ¹³C, and ¹⁵N resonance assignments of Fusarium solani pisi cutinase and preliminary features of the structure in solution

Abstract

Essentially complete (96%) sequence-specific assignments were made for the backbone and side-chain 'H, I3C, and "N resonances of Fusarium solanipisi 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 Ala3' has a strongly upfield-shifted resonance at 3.97 ppm, indicative of an amidearomatic hydrogen bond to the indole ring of Trp69 that stabilizes the N-terminal side of the parallel @-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.Keywords: amide-aromatic hydrogen bond; cutinase; heteronuclear 3D NMR; protein; resonance assignment; secondary structure Fusarium solani pisi cutinase belongs to a group of homologous enzymes capable of degrading cutin (Kolattukudy et al., 1984), the insoluble lipid-polyester matrix covering the surface of plants. Cutinases are produced by several phytopathogenic fungi and pollen, enabling them to gain entry into the plant by enzymatic digestion of its cuticle. Moreover, these enzymes catalyze the hydrolysis of ester bonds of triglycerides (de Geus et al., 1990;Egmond et al., 1994aEgmond et al., , 1994bvan der Hijden et al., 1994). In true lipases, the hydrophobic binding site is buried by a flap region, which supposedly helps to avoid aggregation by hiding the binding site from the solvent. During adsorption to a lipid layer, a significant rearrangement of this flap increases the hydrophobic surface at the lipid-binding region, improving adsorption and allowing binding of the substrate (Brzozowski et al., 1991;Derewenda et al., 1992;van Tilbeurgh et al., 1993). E solanipisi cutinase does not possess a pronounced flap region covering the active site (Martinez et al.,

Thermodynamic and structural investigation of the specific SDS binding of humicola insolens cutinase

“…A similar phenomenon was observed for the structurally similar Pseudomonas mendocina lipase that also exhibited missing signals around the active site at 303 K . However, for that enzyme signals could be observed at 313 K. The NMR spectrum of FsC did not show this feature, as all signals were visible . This means that the intramolecular mobility of the loops is altered in HiC as compared to FsC.…”

“…A similar phenomenon was observed for the structurally similar Pseudomonas mendocina lipase that also exhibited missing signals around the active site at 303 K. 32 However, for that enzyme signals could be observed at 313 K. The NMR spectrum of FsC did not show this feature, as all signals were visible. 33 This means that the intramolecular mobility of the loops is altered in HiC as compared to FsC. The reason for this might be the origin that HiC is from a thermophilic organism, as such proteins generally possess less structural flexibility at room temperature than proteins from mesophiles.…”

De novo design, synthesis and screening of a combinatorial library of complementary ligands directed towards the surface of cutinase from Fusarium solani pisi

“…The relation between structure and function (Martinez et al, 1992;Longhi et al, 1997;Prompers et al, 1997) and the stability (Carvalho et al, 1999) of cutinase from Fusarium solani pisi have been extensively investigated. Immobilization has been explored as an approach for protein stabilization.…”

Fusarium Graminearum spore detection using magnetic resonance force microscopy

“…The calculated force which acts on the cantilever tip results in a deviation in the resonance frequency of the cantilever, which can be measured using an arrangement of laser inferometers and strain gauges. By measuring the change in frequency, it should be possible to begin developing a profile which describes the cell wall chemical makeup of the targeted mold spore, as shown in [10].…”