Biosynthesis and enzymology of fluorometabolite production in Streptomyces cattleya

Abstract

Organofluorine compounds are very rare in nature when compared with other organohalogens and the way by which fluorine is inserted into these compounds was unknown before the investigations described in this thesis. The biochemical processes involved in fluoroacetate and 4-fluorothreonine formation in the actinomycete Streptomyces cattleya have been explored by using isotopic labelling approaches, enzymatic studies and protein purification. The synthesis and feeding of [1-²H₁]-fluoroacetaldehyde to resting cells of S. cattleya proved that fluoroacetaldehyde is a common intermediate in fluorometabolite biosynthesis and is the direct precursor to 4-fluorothreonine. Isotopic labelling studies with [1,1-²H₂]- ethanolamine and [1,1-²H₂]- cysteamine demonstrated that such C-2 units are not involved in the biosynthesis of fluoroacetate and 4-fluorothreonine. Experiments with [2-²H, 2-¹⁸O]-glycerol showed that the C-2 oxygen of glycerol becomes incorporated into both fluorometabolites, whereas the deuterium is lost, probably during the in vivo formation of a carbonyl intermediate during glycolysis. A threonine transaldolase was identified and partially purified from S. cattleya. The enzyme catalyses the formation of 4-fluorothreonine from fluoroacetaldehyde and L-threonine in a pyridoxal phosphate dependent process. Investigations into the mechanism of the transaldolase have been carried out using [1,2,2,2-²H₄]-acetaldehyde and [4,4,4-²H₃]-DL-threonine. The experiments have shown that the threonine transaldolase is a novel enzyme, which is distinct to other enzymes such as threonine aldolase or serine hydroxymethyltransferase, as glycine is not used as a substrate and only L-threonine is accepted. The enzyme responsible for the formation of a C-F bond in S. cattleya was identified and purified to homogeneity. This fluorination enzyme, the first of its class, mediates a reaction between inorganic fluoride ion and S'-adenosyl-L-methionine to generate 5'- fluoro-5'-deoxyadenosine. The fluorinase enzyme was found to be a hexamer with a molecular mass of approximately 180-190 kDa.