Hydrolysis of plant cuticle by plant pathogens. Properties of cutinase I, cutinase II, and a nonspecific esterase isolated from Fusarium solani pisi

Abstract

The properties of the homogeneous cutinase I, cutinase II, and the nonspecific esterase isolated from the extracellular fluid of cutin-grown Fusarium solani F. pisi (R.E. Purdy and P.E. Kolattukudy (1975), Biochemistry, preceding paper in this issue) were investigated. Using tritiated apple cutin as substrate, the two cutinases showed similar substrate concentration dependence, protein concentration dependence, time course profiles, and pH dependence profiles with optimum near 10.0. Using unlabeled cutin, the rate of dihydroxyhexadecanoic acid release from apple fruit cutin by cutinase I was determined to be 4.4 mumol per min per mg. The cutinases hydrolyzed methyl hexadecanoate, cyclohexyl hexadecanoate, and to a much lesser extent hexadecyl hexadecanoate but not 9-hexadecanoyloxyheptadecane, cholesteryl hexadecanoate, or hexadecyl cinnamate. The extent of hydrolysis of these model substrates by cutinase I was at least three times that by cutinase II. The nonspecific esterase hydrolyzed all of the above esters except hexadecyl cinnamate, and did so to a much greater extent than did the cutinases. None of the enzymes hydrolyzed alpha- or beta-glucosides of p-nitrophenol. p-Nitrophenyl esters of fatty acids from C2 through C18 were used as substrates and V's and Kms were determined...

Regulation of Constitutively Expressed and Induced Cutinase Genes by Different Zinc Finger Transcription Factors inFusarium solani f. sp. pisi(Nectria haematococca)

“…Whether the constitutively expressed cutinase and the inducible cutinase are encoded by the same or different genes is not known, and the nature of the transcription factors that are involved in the constitutive expression of cutinase is unknown. Two different but very similar cutinases had been isolated from F. solani pisi (8,9), and the gene previously cloned matched the amino acid sequence of one of these proteins (10), suggesting that another gene encodes the other. Such a gene had not been previously cloned, although Southern analysis had indicated the presence of multiple cutinase genes in the F. solani pisi genome (11).…”

Structural Studies on Cutinase, a Glycoprotein Containing Novel Amino Acids and Glucuronic Acid Amide at the N Terminus

“…Glucosamine and galactosamine were reduced with NaBH4 and the products were desalted using a Dowex-SOW, NHZ column. Cutin was isolated from mature 'Golden delicious' apple fruits and 60 mesh powder was prepared as described previously [2].…”

“…The residue was redissolved in a small volume of H20, dialyzed against H2O overnight and the protein precipitated at 50 % saturation with (NH4)2S04 was collected. Cutinase was purified by Sephadex G-100 gel filtration, followed by ion-exchange chromatography with QAE-Sephadex and SP-Sephadex as described before [2]. Aliquots from each fraction obtained from the latter were lyophilized and checked for carbohydrate content by the phenoljsulfuric acid method [4], using glucose as standard.…”

“…In the case of cutinase 11, the cationic electrophoresis gel showed a single carbohydrate-staining band coincident with the protein-staining band. Upon dodecylsulfate electrophoresis the parent protein of M , 22000 as well as the two fragments (of M , 11000 and 10000) derived by proteolytic nicks [2] were stained by both the carbohydrate and protein stains. Thus cutinase I and cutinase I1 appeared to have covalently attached carbohydrates and, in the case of the latter, carbohydrate residues are present in both halves of the peptide chain.…”

“…A phytopathogenic fungus Fusurium solani f. pisi produces two extracellular isozymes (cutinase I and cutinase 11) which catalyze the hydrolysis of cutin [2]. In view of the observation that extracellular proteins are often glycoproteins we examined the novel polyester-hydrolyzing enzyme, cutinase, for carbohydrates.…”