Abstract
Juvenile hormones have attracted attention as safe and selective targets for the design and development of environmentally friendly and biorational insecticides. In the juvenile hormone III biosynthetic pathway, the enzyme farnesol dehydrogenase catalyzes the oxidation of farnesol to farnesal. In this study, farnesol dehydrogenase was extracted from Polygonum minus leaves and purified 204-fold to apparent homogeneity by ion-exchange chromatography using DEAE-Toyopearl, SP-Toyopearl, and Super-Q Toyopearl, followed by three successive purifications by gel filtration chromatography on a TSK-gel GS3000SW. The enzyme is a heterodimer comprised of subunits with molecular masses of 65 kDa and 70 kDa. The optimum temperature and pH were 35°C and pH 9.5, respectively. Activity was inhibited by sulfhydryl reagents, metal-chelating agents and heavy metal ions. The enzyme utilized both NAD+ and NADP+ as coenzymes with K m values of 0.74 mM and 40 mM, respectively. Trans, trans-farnesol was the preferred substrate for the P. minus farnesol dehydrogenase. Geometrical isomers of trans, trans-farnesol, cis, trans-farnesol and cis, cis-farnesol were also oxidized by the enzyme with lower activity. The K m values for trans, trans-farnesol, cis, trans-farnesol and cis, cis-farnesol appeared to be 0.17 mM, 0.33 mM and 0.42 mM, respectively. The amino acid sequences of 4 tryptic peptides of the enzyme were analyzed by MALDI-TOF/TOF-MS spectrometry, and showed no significant similarity to those of previously reported farnesol dehydrogenases. These results suggest that the purified enzyme is a novel NAD(P)+-dependent farnesol dehydrogenase. The purification and characterization established in the current study will serve as a basis to provide new information for recombinant production of the enzyme. Therefore, recombinant farnesol dehydrogenase may provide a useful molecular tool in manipulating juvenile hormone biosynthesis to generate transgenic plants for pest control.
Highlights
Juvenile hormones (JHs) are a family of sesquiterpenes that play important roles in the development, metamorphosis, reproduction, polyphenism, and behavioral changes of insects [1]
To elucidate the JH III biosynthetic pathway in plant, we investigated enzymes participating in this sesquiterpene metabolism pathway in Polygonum minus
P. minus farnesol dehydrogenase was purified with a high yield (3.2%) by 6 chromatographic steps, including three successive runs on a gel filtration chromatography
Summary
Juvenile hormones (JHs) are a family of sesquiterpenes that play important roles in the development, metamorphosis, reproduction, polyphenism, and behavioral changes of insects [1]. The biosynthetic pathway of juvenile hormone III (JH III, methyl-10R,11-epoxy-3,7,11-trimethyl-2E, 6E-dodecadienoate) is well conserved in insects [7]. JH III and its biosynthetic precursor in insects, methyl farnesoate, were identified in the sedges, Cyperus iria L. and C. aromaticus [19]. Enzyme activities of farnesyl pyrophosphate synthase, farnesyl pyrophatase, farnesol dehydrogenase and methyltransferase which involved in JH III biosynthesis were detected in several plants [11, 21,22,23,24,25,26,27]. More detailed understanding of the enzymes in JH III biosynthetic and metabolic pathways in plant will be useful for the development of new approaches towards integrated pest management using recombinant DNA technology [29] by deployment of the genetically transformed plants for pest control [30]
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