Purification and partial amino acid sequences of an esterase from tomato

Abstract

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 μM and a V max value of 7.97 nkat/mg, an pH optimum of 9.0 and a temperature optimum of 40 °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 α/β hydrolase fold protein superfamily.