Synthesis and characterization of a new cutinase substrate, 4-nitrophenyl (16-methyl sulfone ester) hexadecanoate.
Phytopathogenic fungi penetrate plants by breaking down the cuticular barrier with cutinase. Cutinases are extracellular hydrolytic enzymes that degrade cutin, a polyester composed of hydroxy and epoxy fatty acids. Until now, cutinase has been recognized by its ability to release labeled cutin monomers or by a non-specific esterase assay based on the hydrolysis of p-nitrophenyl esters of short fatty acids. In this work, an insoluble p-nitrophenyl derivative was synthesized and purified, and its structure was determined to be 4-nitrophenyl (16-methyl sulfone ester) hexadecanoate (pNMSEH) by nuclear magnetic resonance (H+ NMR) analysis. pNMSEH was tested as a new cutinase substrate with Pseudomonas mandocino cutinase and porcine liver esterase. While a linear release over time of p-nitrophenol (pNP) was recorded in the presence of cutinase, no response was obtained with the esterase. The calculated kinetic parameters of pNMSEH hydrolysis by cutinase revealed a high specificity (Km=1.8mM), albeit a low catalytic rate (Vmax=10.5 micromol min(-l)l(-1)). This new synthetic substrate may be helpful for detecting and assaying cutinase activity in mixed solutions, such as crude fungal extracellular extracts.
Degani
Ofir
O
Department of Plant Physiology, Faculty of Biology, Technion-Israel Institute of Technology, Haifa 32000, Israel.
Salman
Hussein
H
Gepstein
Shimon
S
Dosoretz
Carlos G
CG
D000818:Animals; D002265:Carboxylic Ester Hydrolases / Q000138:chemical synthesis* / Q000737:chemistry*; D002384:Catalysis; D002851:Chromatography, High Pressure Liquid; D004121:Dimethyl Sulfoxide; D004952:Esters; D007700:Kinetics; D008099:Liver / Q000201:enzymology; D009682:Magnetic Resonance Spectroscopy; D015394:Molecular Structure; D010168:Palmitates / Q000737:chemistry*; D044203:Pseudomonas mendocina / Q000201:enzymology; D013379:Substrate Specificity; D013450:Sulfones / Q000138:chemical synthesis* / Q000737:chemistry* / Q000302:isolation & purification; D013552:Swine
D004952:Esters; D010168:Palmitates; D013450:Sulfones; C025910:dimethyl sulfone; D002265:Carboxylic Ester Hydrolases; C019952:cutinase; D004121:Dimethyl Sulfoxide