Intermediates and enzymes involved in fluorometabolite biosynthesis in Streptomyces cattleya

Abstract

Enzymatic halogenation occurs during the biosynthesis of more than 4,000 natural products. The presence of fluorinated natural products is much less common, with only 13 reported to date. The bacterium Streptomyces cattleya is known to biosynthesise two fluorinated secondary metabolites, fluoroacetate and 4-fluorothreonine. The precursor to these secondary metabolites is known to be fluoroacetaldehyde. It had previously been shown that a fluorination enzyme mediates a reaction between S-adenosyl-L-methionine (SAM) and F to generate 5'-fluoro-5'-deoxyadenosine (5'-FDA). This is the first committed step on the biosynthetic pathway. The pathway between 5' -FDA and fluoroacetaldehyde had not been investigated in detail prior to the work carried out in this thesis. A purine nucleoside phosphorylase has been partially purified from cell-free extracts which catalyses the phosphorolytic cleavage of 5'-FDA to 5-fluoro-5-deoxY-D-ribose-1- phosphate (5-FDRP). Substrate specificity shows a profile which shares a close similarity to bacterial 5'-methylthioadenosine phosphorylases (MTAP's). The identification of a gene cluster encoding enzymes responsible for fluorometabolite biosynthesis shows the PNP gene located adjacent to the fluorinase gene, reinforcing the involvement of this enzyme in the fluorometabolite pathway. It is shown that 5-FDRP is converted to 5-fluoro-5-deoxy-D-ribulose-1-phosphate (5- FDRibP) via an isomerase activity. The enzyme responsible for this transformation has been partially purified from cell free extracts (CFE's). Another metabolite was identified as 5-fluoro-5-deoxY-D-xylulose-l-phosphate (5-FDXyuP), a diastereoisomer of 5-FDRibP, which appears to be an adventitious product in CFE's of S. cattleya. Two DHAP dependent aldolases have been identified, one of which is a putative L- fuculose-1-phosphate aldolase which catalyses conversion of 5-FDRibP to fluoroacetaldehyde. The other, an L-fructose 1,6-bisphosphate aldolase has been purified to homogeneity and catalyses an aldol reaction between DHAP and fluoroacetaldehyde to generate 5-FDXyuP. This enzyme is most probably one of primary metabolism.