Characterization of three cloned and expressed 13-hydroperoxide lyase isoenzymes from alfalfa with unusual N-terminal sequences and different enzyme kinetics

Three full-length cDNAs from alfalfa seedlings coding for hydroperoxide lya ses were cloned and expressed in Escherichia coli and characterized as cyto chrome P450 enzymes. The isoenzymes were specific for 13-hydroperoxy linole ic and linolenic acids and did not use the 9-hydroperoxy isomers as substra tes. Because alfalfa contains both specificities, this indicates the presen ce of two different types of hydroperoxide lyases, each specific for one ki nd of substrate. The enzymes contain 480 amino acids (54 kDa) and contain a n unusual, nonplastidic N-terminal sequence of 22 amino acids, which strong ly reduces the enzyme activity. The only known presequence of a hydroperoxi de lyase (from Arabidopsis thaliana) was considered to be a transit sequenc e. The reduced enzyme activity, however, indicates that the hydroperoxide l yases with N-terminal extensions could be pro-enzymes. This hypothesis is s upported by the fast release of hydroperoxide lyase products by plants upon wounding. One of the isoenzymes showed a strongly decreased V-max and K-m compared to the other two. Because this is probably due to the substitution of Ser377 by Phe; the residue at position 377 seems to be important. This is the first time that sufficient quantities of hydroperoxide lyase have be en obtained for characterization studies, by circumventing difficult purifi cation procedures and degradation of the enzyme. The high expression level, easy purification, good stability and high specificity make these cloned h ydroperoxide lyases excellent tools to study the reaction mechanism and str ucture. We postulate an integrated reaction mechanism, based on the known c hemistry of cytochrome P450 enzymes. This is the first mechanism that unifi es all observed features of hydroperoxide lyases.