In Vitro Reconstituted Biotransformation of 4-Fluorothreonine from Fluoride Ion: Application of the Fluorinase

SummaryIn this paper, we report that fluoride ion is converted to the amino acid/antibiotic 4-fluorothreonine 2 in a biotransformation involving five (steps a–e) overexpressed enzymes. The biotransformation validates the biosynthetic pathway to 4-fluorothreonine in the bacterium Streptomyces cattleya (Schaffrath et al., 2002). To achieve an in vitro biotransformation, the fluorinase and the purine nucleoside phosphorylase (PNP) enzymes (steps a and b), which are coded for by the flA and flB genes of the fluorometabolite gene cluster in S. cattleya, were overexpressed. Also, an isomerase gene product that can convert 5-FDRP 6 to 5-FDRibulP 7 (step c) was identified in S. cattleya, and the enzyme was overexpressed for the biotransformation. A fuculose aldolase gene from S. coelicolor was overexpressed in E. coli and was used as a surrogate aldolase (step d) in these experiments. To complete the complement of enzymes, an ORF coding the PLP-dependent transaldolase, the final enzyme of the fluorometabolite pathway, was identified in genomic DNA by a reverse genetics approach, and the S. cattleya gene/enzyme was then overexpressed in S. lividans. This latter enzyme is an unusual PLP-dependent catalyst with some homology to both bacterial serine hydroxymethyl transferases (SHMT) and C5 sugar isomerases/epimerases. The biotransformation demonstrates the power of the fluorinase to initiate C-F bond formation for organo-fluorine synthesis

An enzymatic Finkelstein reaction : fluorinase catalyses direct halogen exchange

We thank the Engineering and Physical Sciences Research Council, UK, for a research grant.The fluorinase enzyme from Streptomyces cattleya is shown to catalyse a direct displacement of bromide and iodide by fluoride ion from 5′-bromodeoxyadenosine (5′-BrDA) and 5′-iododeoxyadenosine (5′-IDA) respectively to form 5′-fluorodeoxyadenosine (5′-FDA) in the absence of L-methionine (L-Met) or S-adenosyl-L-methionine (SAM). 5′-BrDA is the most efficient substrate for this enzyme catalysed Finkelstein reaction.PostprintPeer reviewe

The Gene Cluster for Fluorometabolite Biosynthesis in Streptomyces cattleya: A Thioesterase Confers Resistance to Fluoroacetyl-Coenzyme A

SummaryA genomic library of Streptomyces cattleya was screened to isolate a gene cluster encoding enzymes responsible for the production of fluorine-containing metabolites. In addition to the previously described fluorinase FlA which catalyzes the formation of 5′-fluoro-5′-deoxyadenosine from S-adenosylmethionine and fluoride, 11 other putative open reading frames have been identified. Three of the proteins encoded by these genes have been characterized. FlB was determined to be the second enzyme in the pathway, catalyzing the phosphorolytic cleavage of 5′-fluoro-5′-deoxyadenosine to produce 5-fluoro-5-deoxy-D-ribose-1-phosphate. The enzyme FlI was found to be an S-adenosylhomocysteine hydrolase, which may act to relieve S-adenosylhomocysteine inhibition of the fluorinase. Finally, flK encodes a thioesterase which catalyzes the selective breakdown of fluoroacetyl-CoA but not acetyl-CoA, suggesting that it provides the producing strain with a mechanism for resistance to fluoroacetate

Studies and application of the enzymes of fluorometabolite biosynthesis in Streptomyces cattleya

This thesis focuses on studies investigating the structure of intermediates involved in fluorometabolite biosynthesis, and the potential applications of the fluorinase enzyme in positron emission tomography (PET). Chapter 1 introduces the rare natural occurrence of fluorinated compounds. The bacterium Streptomyces cattleya is known to biosynthesise two fluorinated secondary metabolites: the toxin fluoroacetate (FAc) and the antibiotic 4-fluorothreonine (4-FT). The enzymes and intermediates identified on this fluorometabolite biosynthetic pathway in S. cattleya, prior to this research, are discussed in detail. Chapter 2 presents studies towards the unambiguous structural identification of (3R,4S)-5- deoxy-5-fluoro-D-ribulose-1-phosphate (5-FRulP) as the third fluorinated intermediate on the biosynthetic pathway to fluoroacetate and 4-fluorothreonine in S. cattleya. Chapter 3 describes the synthetic routes to key molecules, necessary as reference compounds and substrates, to underpin the subsequent studies in this thesis. In particular, synthetic routes to 5'-deoxy-5'-fluoroadenosine (5'-FDA), 5'-deoxy-5'-fluoroinosine (5'-FDI), 5-deoxy-5-fluoro-D-ribose (5-FDR) and 5-deoxy-5-fluoro-D-xylose (5-FDX) are described. Chapter 4 describes the use of the fluorinase enzyme from S. cattleya as a tool for the synthesis of new [¹⁸F]-labelled sugars with potential application in positron emission tomography (PET). A new route to 5-deoxy-5-[¹⁸F]fluoro-D-ribose ([¹⁸F]FDR) is developed in a two-step enzymatic synthesis. A total of three potential radiotracers ([¹⁸F]FDA, [¹⁸F]FDR and [¹⁸F]FDI) are synthesised using fluorinase-coupled enzyme reactions. In addition, in vitro studies are reported with these [¹⁸F]-labelled sugars to investigate their uptake and potential as PET radiotracers in cancer cells. A preliminary rat imaging study with [¹⁸F]FDA is reported. Chapter 5 details the experimental procedures for the compounds synthesised in this research and the biological procedures for chemo-enzymatic syntheses and protein purification

Oligomerization engineering of the fluorinase enzyme leads to an active trimer that supports synthesis of fluorometabolites in vitro

This work was funded by The Novo Nordisk Foundation grant to the Center for Biosustainability (NNF10CC1016517). P.I.N. was funded by grants from The Novo Nordisk Foundation (NNF20CC0035580, and LiFe, NNF18OC0034818), the European Union’s Horizon 2020 Research and Innovation Programme under grant agreement No. 814418 (SinFonia) and the Danish Council for Independent Research (SWEET, DFF-Research Project 8021-00039B). T.K. and M.N.D. were funded by fellowships from the European Union's Horizon 2020 research and innovation program under a Marie Skłodowska Curie project under grant agreement No. 713683 (COFUNDfellowsDTU).The fluorinase enzyme represents the only biological mechanism capable of forming stable C–F bonds characterized in nature thus far, offering a biotechnological route to the biosynthesis of value-added organofluorines. The fluorinase is known to operate in a hexameric form, but the consequence(s) of the oligomerization status on the enzyme activity and its catalytic properties remain largely unknown. In this work, this aspect was explored by rationally engineering trimeric fluorinase variants that retained the same catalytic rate as the wild-type enzyme. These results ruled out hexamerization as a requisite for the fluorination activity. The Michaelis constant (KM) for S-adenosyl-l-methionine, one of the substrates of the fluorinase, increased by two orders of magnitude upon hexamer disruption. Such a shift in S-adenosyl-l-methionine affinity points to a long-range effect of hexamerization on substrate binding – likely decreasing substrate dissociation and release from the active site. A practical application of trimeric fluorinase is illustrated by establishing in vitro fluorometabolite synthesis in a bacterial cell-free system.Publisher PDFPeer reviewe

Peculiarities of promiscuous l-threonine transaldolases for enantioselective synthesis of β-hydroxy-α-amino acids

Funding This study was funded by Biotechnology and Biological Sciences Research Council UK (BBSRC) (BB/P00380X/1, BB/R00479X/1 and BB/R50547X/1), Scottish Funding Council COVID-19 Grant extension and Bridging Fund, and Industrial Biotechnology Innovation Centre (IBioIC, Scotland). open access via springer agreementPeer reviewedPublisher PD

Exploration of a potential difluoromethyl-nucleoside substrate with the fluorinase enzyme

The authors thank EPSRC and the Scottish Imaging Network (SINAPSE) for grants. DO’H thanks the Royal Society for a Wolfson Research Merit Award and ST is grateful to the John and Kathleen Watson Scholarship for financial support.The investigation of a difluoromethyl-bearing nucleoside with the fluorinase enzyme is described. 5’,5’–Difluoro-5’-deoxyadenosine 7 (F2DA) was synthesised from adenosine, and found to bind to the fluorinase enzyme by isothermal titration calorimetry with similar affinity compared to 5’–fluoro-5’-deoxyadenosine 2 (FDA), the natural product of the enzymatic reaction. F2DA 7 was found, however, not to undergo the enzyme catalysed reaction with l–selenomethionine, unlike FDA 2, which undergoes reaction with l-selenomethionine to generate Se-adenosylselenomethionine. A co-crystal structure of the fluorinase and F2DA 7 and tartrate was solved to 1.8 Å, and revealed that the difluoromethyl group bridges interactions known to be essential for activation of fluoride for reaction. An unusual hydrogen bonding interaction between the hydrogen of the difluoromethyl group and one of the hydroxyl oxygens of the tartrate ligand was also observed. The bridging interactions, coupled with the inherently stronger C–F bond in the difluoromethyl group, offers an explanation for why no reaction is observed.PostprintPeer reviewe

An unusual metal-bound 4-fluorothreonine transaldolase from Streptomyces sp. MA37 catalyses promiscuous transaldol reactions

Open Access via the Springer Compact Agreement. This study was funded by IBioIC PhD studentship (LW), Leverhulme Trust Research Project (HD and MHT, project No. RPG-2014-418), The Elphinstone Scholarship of University of Aberdeen (QF), Leverhulme Trust-Royal Society Africa award (KK and HD, AA090088) and the jointly funded UK Medical Research Council – UK Department for International Development (MRC/DFID) Concordat agreement African Research Leaders Award (KK and HD, MR/S00520X/1), Biotechnology and Biological Sciences Research Council UK (HD and SW, BB/P00380X/1) and National Natural Science Foundation of China (31,570,033, 31,811,530,299, and 31,870,035 to YY), and the Royal Society-NSFC Newton Mobility Grant Award (IEC\NSFC\170,617 to HD and YY).Peer reviewedPublisher PD

Fluorometabolite biosynthesis : isotopically labelled glycerol incorporations into the antibiotic nucleocidin in Streptomyces calvus

Deuterium and carbon-13 labelled glycerols have been fed to Streptomyces calvus fermentations and isotope incorporation into the fluorine containing antibiotic nucleocidin have been evaluated by 19F-NMR. A single deuterium atom was incorporated from [2H5]- and (R)-[2H2]- glycerol into C-5’ of the antibiotic, suggesting that an oxidation occurs at this carbon after ribose ring assembly from glycerol (pentose phosphate pathway), during nucleocidin biosynthesis.PostprintPostprintPeer reviewe

6-De­oxy-6-fluoro-d-galactose

The crystal structure unequivocally confirms the relative stereochemistry of the title compound, C6H11FO5. The absolute stereochemistry was determined by the use of d-galactose as the starting material. The compound exists as a three-dimensional O—H⋯O hydrogen-bonded network with each mol­ecule acting as a donor and acceptor for four hydrogen bonds