Elucidation of cutinase structure-activity relationships
Fungal organisms produce extracellular enzymes called cutinases. In nature, cutinases catalyze hydrolysis of cutin ester-bonds. Cutin is a lipid-like polyester found in the cuticle of higher plants that serves as an armor protecting plant cells from microbial attack (Figs. 11 and 12). Thus, organisms that excrete cutinases have a distinct advantage in gaining entry into plant leaves leading to further attack on plant systems.
Figure 11. Major cutin monomer structures. X= 5, 6, 7, 8; Y= 5, 6, 7, 8, X+Y= 13. Minor monomers such as fatty acids, fatty alcohols, aldehydes, ketones, diacids as well as hydroxycinnamic acids can also be liberated.
Cutinases are the smallest members of the serine α/β hydrolase superfamily, with molecular weights about 20 K. The cutinase from Fusarium solanii has an active site consisting of the catalytic triad Ser120, Asp 175 and His188. However, unlike most lipases, the catalytic serine is not buried under an amphipathic loop but is accessible to the solvent. The ability of cutinase to hydrolyze this natural polyester begs the question as to its activity on other natural and synthetic substrates with ester linkages.
Figure 12. Side view SEM of tomato fruit cuticle showing the epicuticular surface and cutin biopolyester.
Scheme 11. Structures of PET and PVAc, synthetic polymer substrates hydrolyzed by cutinases
Comparative studies of different cutinases are lacking. Our group, in collaboration with that of Prof. Montclare (NYU-POLY) have initiated systematic studies with different cutinases to characterize their structure-activity relationships. Through this work we hope to ascertain what structural characteristics lead to cutinases with exceptional activity for reactions of interest that include polymer modifications and cutin hydrolysis. Ultimately, information gained will be used to provide a rational basis for engineering cutinases with improved activity and stability.
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Work was initiated by selecting cutinases from a pdb and genbank search and by pair-wise alignment of sequences relative to Fusarium solani cutinase (FsC). The selection criteria were cutinases that exhibited a sequence identity between 45 and 65% over a minimum overlap of 175 amino acids. The result was the selection of cutinase sequences produced by Humicola insolens (HiC), Alternaria brassicicola (AbC), Aspergillus fumigatus (AfC) and Aspergillus oryzae (AoC). All these species are involved in the destruction of valuable plants and crops with varied types and degrees of pathogenicity. The cutinases from the above set were PCR assembled from synthetic oligonucleotides, cloned and transformed into Escherichia coli. The resulting plasmids were sequenced and then transformed into P. pastoris. HiC, AbC, AfC, AoC and FsC were successfully synthesized and purified by FPLC. Studies on these cutinases are focused on establishing sequence-structure-activity relationships using substrates that include naturally derived cutin, cutin-related synthetic polyester structures and industrial important polymers including poly(ethylene terephthalate) and polyvinyl acetate (see Scheme 11). Extraordinary activities of cutinases have already been established. For example, they are powerful hydrolases that convert PET to their corresponding monomers. Results of three manuscripts describing work accomplished thus far is currently in review.