Cytochrome P450s are a ubiquitous family of enzymes, having been found in organisms ranging in complexity from prokaryotes eg. bacteria through to eukaryotes eg. plants and mammals. The primary function of a P450 is the oxidation of substances either (1) to detoxify these compounds and initiate their removal from the organism, (2) to begin the degradation of compounds for use as a carbon sources or (3) as en2ymes involved in biosynthetic pathways. There were several disparate aims to this project. The first was to determine the role of a biosynthetic P450 found in Bacillus subtilis. The second was to determine the regio and stereochemical properties of the enzyme which catalyses the penultimate step in the biosynthesis of a proposed pheromone from Bactrocera (fruit fly). The third aim was to gain information on the P450s present in B. cacuminata in order to facilitate the isolation of the enzyme responsible for this penultimate step.
A P450 gene from B. subtilis, bioI, is located in an operon responsible for the biosynthesis of biotin. BioI has previously been cloned, ejq)ressed and purified and initial characterization revealed some unusual properties. These properties have subsequently been found to be the result of the expressed P450, BioI being complexed b acyl carrier protein (ACP), a protein involved in fatty acid biosynthesis. The origin of pimeUc acid, a dicarboxylic acid precursor of biotin, is not known in B. subtilis and it was suggested that BioI may be involved in the biosynthesis of this molecule. As the BioI-ACP complex isolated was found to be loaded with a range of fatty acids, a number of fatty acids were tested as potential substrates for Biol. It was discovered, after finding suitable redox partners in cindoxin and E. coli flavodoxin, that BioI catalysed the cleavage of a long chain fatty acid (14-18C) to produce pimelic acid.
It had previously been established that the penultimate step in the biosynthesis of 1,7- dioxaspiro[5.5]undecane, a pheromone from Bactrocera oleae (oUve fly), is the hydroxylation of 6-«-butyl-tetiahydro-pyran-2-ol. To determine whether this was a general step in the biosynthesis of this molecule and other similar spiroacetals in Bactrocera fruit flies, two Australian Bactrocera sp. were investigated It was determined that both B. cacuminata and B. cucumis utilize this penultimate hydroxylation to produce 1,7- dioxaspiro[5.5]undecane and 2,8-dimethyl-l,7-dioxaspiro[5.5] undecane, respectively, by the oxidation four carbons from the hemiketal center. The specificity of the hydroxylase which catalyses this reaction were also investigated.
P450s are responsible for the majority of hydroxylations that occur in nature. It was therefore postulated that the penultimate hydroxylation, involved in the formation of 1,7- dioxaspiro[5.5]undecane in B. cacuminata, was catalysed by a P450. Other enzymes that perform these reactions include the nonhaem iron oxygenases which are significantly less common than P450s. Work was undertaken to locate this P450, as elucidation of the biosynthesis of this proposed pheromone may be useful for the design of 'biologically friendly' insecticides. A cDNA library of male B. cacuminata rectal glands was constructed The screening of this library with short (18-23bp), degenerate, oligonucleotide probes, based on the conserved cysteine region of two P450 families (CYP4 and CYP6), failed to uncover any P450s. PCR was then employed to amplify fragments of P450 genes using primers designed to two conserved CYP4 regions; the I-helix and the conserved cysteine region When the cDNA obtained from the abdomens of male B. cacuminata was used as the template, this PCR produced several partial P450 fragments (^prox. 450bp). This information may be useful in locating the P450 of interest .