Mechanistic studies on enzymes of secondary metabolism : 5'-fluoro-5'-deoxyadenosine synthase and 1-deoxy-D-xylulose-5-phosphate reductoisomerase

Abstract

A fluorination enzyme (fluorinase) has recently been isolated in St. Andrews from the bacterium Streptomyces cattleya. This enzyme mediates a reaction between fluoride ion and Sadenosyl-L-methionine (SAM) to generate 5'-fluoro-5'-deoxy-adenosine (5'-FDA). In this thesis the stereochemical course of biological fluorination has been evaluated. This involved the preparation of a stereospecifically labelled sample of ATP {(5'R)-[²H₁]-ATP}. This substrate generated a stereospecifically labelled sample of 5'-FDA after a coupled enzyme reaction involving the SAM synthase and then the fluorinase. Evaluation of the absolute configuration of the chiral fluoromethyl group at the 5'-carbon of 5'-FDA (HDFC-) by ²H-NMR analysis in a chiral liquid-crystalline medium, and by comparison with a stereospecifically labelled sample of (5'S)-[²H₁]-FDA, established that the fluorinase catalyses fluorination with an inversion of configuration, consistent with an Sₙ2 reaction mechanism. This is discussed in detail in chapter 4.

An investigation into halide binding prior to nucleophilic substitution of SAM was also addressed. This involved the preparation of a sample of 9-(β-D-erytho-furanosyl)adenosine a compound which was co-crystallised with the fluorinase. An enzyme crystal structure with 9-(β-D-eythro-furanosyl)adenosine was obtained, although the chloride ion was not evident. This is discussed in chapter 3. The DXR is an enzyme on the non-mevalonate pathway to isopentenyl diphosphate and dimethylallyl diphosphate that catalyses the conversion of 1-deoxy-D-xylulose 5-phosphate (DXP) to 2-C-methyl-D-erythritol 4-phosphate (MEP). A candidate inhibitor was designed and prepared to explore the mechanism of this enzyme. This is discussed in detail in chapter 6.