Christoph Schaffrath

Biochemistry: Biosynthesis of an organofluorine molecule

Although fluorine in the form of fluoride minerals is the most abundant halogen in the Earths crust, only 12 naturally occurring organofluorine compounds have so far been found, and how these are biosynthesized remains a mystery. Here we describe an enzymatic reaction that occurs in the bacterium Streptomyces cattleya and which catalyses the conversion of fluoride ion and S-adenosylmethionine (SAM) to 5′-fluoro-5′-deoxyfluoroadenosine (5′-FDA). To our knowledge, this is the first fluorinase enzyme to be identified, a discovery that opens up a new biotechnological opportunity for the preparation of organofluorine compounds.

Crystal structure and mechanism of a bacterial fluorinating enzyme

Fluorine is the thirteenth most abundant element in the earths crust, but fluoride concentrations in surface water are low and fluorinated metabolites are extremely rare. The fluoride ion is a potent nucleophile in its desolvated state, but is tightly hydrated in water and effectively inert. Low availability and a lack of chemical reactivity have largely excluded fluoride from biochemistry: in particular, fluorines high redox potential precludes the haloperoxidase-type mechanism used in the metabolic incorporation of chloride and bromide ions. But fluorinated chemicals are growing in industrial importance, with applications in pharmaceuticals, agrochemicals and materials products. Reactive fluorination reagents requiring specialist process technologies are needed in industry and, although biological catalysts for these processes are highly sought after, only one enzyme that can convert fluoride to organic fluorine has been described. Streptomyces cattleya can form carbon–fluorine bonds and must therefore have evolved an enzyme able to overcome the chemical challenges of using aqueous fluoride. Here we report the sequence and three-dimensional structure of the first native fluorination enzyme, 5′-fluoro-5′-deoxyadenosine synthase, from this organism. Both substrate and products have been observed bound to the enzyme, enabling us to propose a nucleophilic substitution mechanism for this biological fluorination reaction.

Isolation and characterisation of 5'-fluorodeoxyadenosine synthase, a fluorination enzyme from Streptomyces cattleya.

5′‐Fluorodeoxyadenosine synthase, a C–F bond‐forming enzyme, has been purified from Streptomyces cattleya. The enzyme mediates a reaction between inorganic fluoride and S‐adenosyl‐L‐methionine (SAM) to generate 5′‐fluoro‐5′‐deoxyadenosine. The molecular weight of the monomeric protein is shown to be 32.2 kDa by electrospray mass spectrometry. The kinetic parameters for SAM (K m 0.42 mM, V max 1.28 U/mg) and fluoride ion (K m 8.56 mM, V max 1.59 U/mg) have been evaluated. Both S‐adenosyl‐L‐homocysteine (SAH) and sinefungin were explored as inhibitors of the enzyme. SAH emerged as a potent competitive inhibitor (K i 29 μM) whereas sinefungin was only weakly inhibitory.

Fluorinated natural products: the biosynthesis of fluoroacetate and 4-fluorothreonine in Streptomyces cattleya.

Organofluorine compounds are rare in Nature, with only a handful known to be produced by some species of plant and two microorganisms. Consequently, the mechanism of enzymatic carbon-fluorine bond formation is poorly understood. The bacterium Streptomyces cattleya biosynthesises fluoroacetate and 4-fluorothreonine as secondary metabolites and is a convenient system to study the biosynthesis and enzymology of fluorometabolite production. Using stable-isotope labelled precursors it has been shown that there is a common intermediate in the biosynthesis of the fluorometabolites, which has recently been identified as fluoroacetaldehyde. Studies with cell-free extracts of S. cattleya have identified two enzymes, an aldehyde dehydrogenase and a threonine transaldolase, that are involved in the biotransformation of fluoroacetaldehyde to fluoroacetate and 4-fluorothreonine.

Crystallization and X-ray diffraction of 5'-fluoro-5'-deoxyadenosine synthase, a fluorination enzyme from Streptomyces cattleya

Organofluorine compounds are widely prepared throughout the chemicals industry, but their prepararion generally requires harsh fluorinating reagents and non-aqueous solvents. On the other hand, biology has hardly exploited organofluorine compounds. A very few organisms synthesize organofluorine metabolites, suggesting they have evolved a mechanism to overcome the kinetic desolvation barrier to utilizing F(-)(aq). Here, the purification and crystallization of an enzyme from Streptomyces cattleya which is responsible for the synthesis of the C-F bond during fluoroacetate and 4-fluorothreonine biosynthesis is reported. The protein crystallizes in space group C222(1), with unit-cell parameters a = 75.9, b = 130.3, c = 183.4 A, alpha = beta = gamma = 90 degrees. Data were recorded to 1.9 A at the ESRF. The structure of the protein should provide important insights into the biochemical process of C-F bond formation.

Biosynthesis of fluoroacetate and 4-fluorothreonine in Streptomyces cattleya. Incorporation of oxygen-18 from [2-2H,2-18O]-glycerol and the role of serine metabolites in fluoroacetaldehyde biosynthesis

A series of isotope labelling experiments was carried out to investigate the biosynthesis of fluoroacetate and 4-fluorothreonine in resting cells of Streptomyces cattleya. Previous studies have shown that fluoroacetaldehyde is a precursor to both of these metabolites and the experiments were conducted to explore in greater detail the metabolic origin of fluoroacetaldehyde in S. cattleya. Ethanolamine and cysteamine are C2 metabolites of serine and cysteine respectively and these two metabolites emerged as candidate precursors to fluoroacetaldehyde in S. cattleya. However feeding experiments with [1,1-2H2]-ethanolamine and [1,1-2H2]-cysteamine did not indicate incorporation into the fluorometabolites, suggesting that these compounds are not relevant precursors to fluoroacetaldehyde in S. cattleya. Upon feeding [2-2H,2-18O]-glycerol to resting cells of S. cattleya, the deuterium atom was not incorporated into 4-fluorothreonine, however the oxygen-18 atom became incorporated into the carboxylate group of fluoroacetate and into the C(3)-O oxygen atom of 4-fluorothreonine. This observation indicates that there is an oxidation at C-2 of glycerol, but that the oxygen atom is formally retained from glycerol during the biosynthesis. In overview, the data suggest that fluoroacetaldehyde is derived from a C3 glycolytic intermediate rather than a C2 amino acid metabolite.