The synthesis and evaluation of natural/unnatural substrates of lepidopteran prenyltransferase
Abstract
Juvenile hormone (JH) is vital to normal insect growth and development. In the lepidopteran species of Manduca Sexta, JH consists of five different methyl epoxy-farnesoate homologs, which are produced via the terpenoid biosynthetic pathway. Prenyltransferase, which catalyzes the formation of the JH carbon backbone, is a key enzyme of this pathway since it may be partially responsible for control of JH homolog production. This research was concerned with the optimization and characterization of lepidopteran prenyltransferase activity in M. Sexta larval corpora allata. Substrate specificity studies were performed that involved the examination of all possible natural and unnatural methyl/ethyl coupling patterns of dimethylallyl diphosphate and geranyl diphosphate. This required the synthesis of all possible natural and unnatural methyl/ethyl homologs of dimethylallyl diphosphate and geranyl diphosphate, as well as those of farnesol. Optimum prenyltransferase activity was obtained with corpora allata homogenates containing 2% Triton X-100, 0.15% BSA, 25% glycerol (all w/v), 2.5 mM MgCl$\sb2$, 10 mM KF, and 0.5 mM mercaptoethanol. Detergents usually do not increase short-chained prenyltransferase activity; however, this system displayed significant enhancement of enzymatic activity in the presence of non-ionic detergents. M. Sexta prenyltransferase activity required divalent cations and had a broad pH maxima. Enzyme localization studies determined that the protein is cytosolic. K$\sb{\rm M}$ values of 6.2 and 3.0 $\mu$M were determined for GPP and IPP, respectively. Enzymatic activity was inhibited by phosphate, diphosphate, NEM, and GGPP. The homologs of geranyl diphosphate were constructed in a convergent manner, by olefination of the corresponding trisnoraldehydes, obtained from terminal oxidative cleavage of geraniol, higher-order vinyl cuprate conjugate addition to acrolein, or cuprate conjugate addition to ethyl 2-pentynoate. To serve as authentic standards, farnesol homologs were prepared by barium-stabilized allylic coupling of the geranyl derivatives to either C-5 or C-6 allylic bromides. Substrate specificity studies using the prepared DMAPP and GPP homologs found that lepidopteran prenyltransferase differs from other short-chain prenyltransferases since it readily accepts ethyl-containing homologs while other enzymes do not.
Degree
Ph.D.
Advisors
Sen, Purdue University.
Subject Area
Organic chemistry|Biochemistry|Agricultural chemicals|Anatomy & physiology|Animals
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