Affinity purification and characterization of a cutinase from the fungal plant pathogen Monilinia fructicola (Wint.) honey

Trifluoromethyl ketones (TFK) are potent inhibitors of a variety of serine hydrolases. The TFK inhibitor, 3-(4-mercaptobutylthio)-1,1,1-trifluoro-2-pr opanone (MBTFP), was found to competitively inhibit cutinase activity (I-50 = 9.4 x 10(-3)) from the fungal plant pathogen Monilinia Fucticola and to serve as an effective affinity ligand for the purification of cutinases fro m culture filtrates. The TFK inhibitors, 3-n-octylthio-1,1,1 -trifluoro-2-p ropanone (OTFP) and 3-n-pentylthio-1,1,1-trifluoro-2-propanone (PTFP), also inhibited cutinase activity with I-50 values of 1.6 x 10(-6) and 2.3 x 10( -4) M, respectively. Buffer containing OTFP was the strongest eluant for cu tinases of M. fructicola and provided the best purification factor and yiel d, although buffers containing OTFP, detergent, and salt were found to be e ffective for eluting cutinases bound to MBTFP-Sepharose. Buffer containing 0.5% Triton X-100 also selectively eluted cutinases from the affinity colum n. Two-dimensional electrophoretic analysis by SDS-PAGE and isoelectric foc using of the affinity-purified cutinase fraction indicated activity associa ted with proteins of pi 8.2 and molecular masses of approximately 18.6 and 20.8 kDa. These proteins hydrolyzed [H-3]cutin and artificial substrates su ch as p-nitrophenylbutyrate and related esters, typical of other cutinases, but differ from previously characterized cutinases in molecular mass. The two low-molecular-weight proteins resolved by 2-D gel electrophoresis were subjected to in-gel digestion with Lys-C and the resulting peptide fragment s were separated by Microbore-HPLC. The amino acid sequences of several int ernal peptide fragments had high homology with cutinase sequences from othe r fungi, particularly the plant pathogen Botrytis cinerea. Our study illust rates the potential of TFK ligands for the affinity purification of cutinas es and indicates that the cutinases from M. fructicola have novel features warranting further study. (C) 2000 Academic Press.