Fluorometabolite biosynthesis in streptomyces cattleya

Abstract

Nature has evolved the ability to form a C-F bond, as exemplified by the bacterium Streptomyces cattleya, which elaborates fluoroacetate (FAc) and 4-fluorothreomne (4- FT). The mechanism of this bond formation are unknown. This thesis probes the biosynthesis of fluoroacetate and 4-fiuorothreonine and in doing so explores the C-F bond forming process. Feeding stable isotope enriched primary metabolites to S. cattleya, followed by (^19)F NMR and GCMS analysis of the resultant fluorometabolites, highlights the role of the glycolytic pathway in delivering a substrate for fluorination. 3-Fluoro-l- hydroxypropan-2-one was synthesised and feeding studies eliminate this as the initial product of fluorination. Fluoroacetaldehyde was identified as a common fluorinated intermediate in the biosynthesis of both FAc and 4-FT. Whole cell studies demonstrate the rapid oxidation of fluoroacetaldehyde to FAc. 4-FT is produced in low quantities by S. cattleya incubated with fluoroacetaldehyde. The synthesis and feeding of [1-(^2)H]- fluoroacetaldehyde provide evidence that the resultant 4-FT is biosynthesised from fluoroacetaldehyde. The biotransformation from fluoroacetaldehyde to FAc was shown in cell free studies to be mediated by an aldehyde dehydrogenase, requiring NAD(^4) as a co-factor. The substrate specificity of fluoroacetaldehyde dehydrogenase was probed by spectrophotometrically monitoring the production of NADH in the presence of different aldehydes. Further cell free experiments probed the biosynthetic origins of fluoroacetaldehyde. Glycolaldehyde phosphate and various phosphorylated glycolytic intermediates were incubated with cell free extracts of S. cattleya and a plethora of co-factors. In the absence of observing fluorination activity, it was shown that the cell free extract acts to dephosphorylate the substrates. The putative role of glycolaldehyde phosphate was explored by feeding isotopically labelled glycolaldehydes to whole cells of the bacterium. The results were not consistent with direct conversion from glycolaldehyde phosphate to fluoroacetaldehyde.