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.
Type
Thesis, PhD Doctor of Philosophy
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