Purification and partial amino acid sequences of an esterase from tomato.
Screening of 18 suspension plant cell cultures of taxonomically distant species revealed that a methyl jasmonate hydrolysing enzyme activity (0.21-5.67 pkat/mg) occurs in all species so far analysed. The methyl jasmonate hydrolysing esterase was purified from cell cultures of Lycopersicon esculentum using a five-step procedure including anion-exchange chromatography, gel-filtration and chromatography on hydroxylapatite. The esterase was purified 767-fold to give an almost homogenous protein in a yield of 2.2%. The native enzyme exhibited a M(r) of 26 kDa (gel-filtration chromatography), which was similar to the M(r) determined by SDS-PAGE and MALDI-TOF analysis (M(r) of 28547 kDa). Enzyme kinetics revealed a K(m) value of 15 microM and a V(max) value of 7.97 nkat/mg, an pH optimum of 9.0 and a temperature optimum of 40 degrees C. The enzyme also efficiently hydrolyzed methyl esters of abscisic acid, indole-3-acetic acid, and fatty acids. In contrast, methyl esters of salicylic acid, benzoic acid and cinnamic acid were only poor substrates for the enzyme. N-Methylmaleimide, iodacetamide, bestatin and pepstatin (inhibitors of thiol-, metal- and carboxyproteases, respectively) did not inactivate the enzyme while a serine protease inhibitor, phenylmethylsulfonyl fluoride, at a concentration of 5 mM led to irreversible and complete inhibition of enzyme activity. Proteolysis of the pure enzyme with endoproteinase LysC revealed three peptide fragments with 11-14 amino acids. N-Terminal sequencing yielded an additional peptide fragment with 10 amino acids. Sequence alignment of these fragments showed high homologies to certain plant esterases and hydroxynitrile lyases that belong to the alpha/beta hydrolase fold protein superfamily.
Stuhlfelder
Christiane
C
Julius-von-Sachs-Institute for Biosciences, Pharmaceutical Biology, University of Würzburg, Julius-von-Sachs-Platz 2, Germany.
Lottspeich
Friedrich
F
Mueller
Martin J
MJ
D000085:Acetates / Q000378:metabolism*; D000595:Amino Acid Sequence; D018929:Cell Culture Techniques; D003517:Cyclopentanes / Q000378:metabolism*; D004950:Esterases / Q000737:chemistry / Q000302:isolation & purification*; D018551:Solanum lycopersicum / Q000201:enzymology*; D008969:Molecular Sequence Data; D054883:Oxylipins; D020539:Sequence Analysis, Protein; D017386:Sequence Homology, Amino Acid
D000085:Acetates; D003517:Cyclopentanes; D054883:Oxylipins; C072239:methyl jasmonate; D004950:Esterases