Global analysis of adenylate-forming enzymes reveals ß-lactone biosynthesis pathway in pathogenic Nocardia.

Enzymes that cleave ATP to activate carboxylic acids play essential roles in primary and secondary metabolism in all domains of life. Class I adenylate-forming enzymes share a conserved structural fold but act on a wide range of substrates to catalyze reactions involved in bioluminescence, nonribosomal peptide biosynthesis, fatty acid activation, and ß-lactone formation. Despite their metabolic importance, the substrates and functions of the vast majority of adenylate-forming enzymes are unknown without tools available to accurately predict them. Given the crucial roles of adenylate-forming enzymes in biosynthesis, this also severely limits our ability to predict natural product structures from biosynthetic gene clusters. Here we used machine learning to predict adenylate-forming enzyme function and substrate specificity from protein sequences. We built a web-based predictive tool and used it to comprehensively map the biochemical diversity of adenylate-forming enzymes across >50,000 candidate biosynthetic gene clusters in bacterial, fungal, and plant genomes. Ancestral phylogenetic reconstruction and sequence similarity networking of enzymes from these clusters suggested divergent evolution of the adenylate-forming superfamily from a core enzyme scaffold most related to contemporary CoA ligases toward more specialized functions including ß-lactone synthetases. Our classifier predicted ß-lactone synthetases in uncharacterized biosynthetic gene clusters conserved in >90 different strains of Nocardia. To test our prediction, we purified a candidate ß-lactone synthetase from Nocardia brasiliensis and reconstituted the biosynthetic pathway in vitro to link the gene cluster to the ß-lactone natural product, nocardiolactone. We anticipate that our machine learning approach will aid in functional classification of enzymes and advance natural product discovery.

Influence of Water and Enzyme on the Post-Transition State Bifurcation of NgnD-Catalyzed Ambimodal [6+4]/[4+2] Cycloaddition.

The enzyme NgnD catalyzes an ambimodal cycloaddition that bifurcates to [6+4]- and [4+2]-adducts. Both products have been isolated in experiments, but it remains unknown how enzyme and water influence the bifurcation selectivity at the femtosecond time scale. Here, we study the impact of water and enzyme on the post-transition state bifurcation of NgnD-catalyzed [6+4]/[4+2] cycloaddition by integrating quantum mechanics/molecular mechanics quasiclassical dynamics simulations and biochemical assays. The ratio of [6+4]/[4+2] products significantly differs in the gas phase, water, and enzyme. Biochemical assays were employed to validate computational predictions. The study informs how water and enzyme affect the bifurcation selectivity through perturbation of the reaction dynamics in the femtosecond time scale, revealing the fundamental roles of condensed media in dynamically controlling the chemical selectivity for biosynthetic reactions.

Production of the Carboxylate Reductase from Nocardia otitidiscaviarum in a Soluble, Active Form for in†vitro Applications.

Accessing aldehydes from carboxylate moieties is often a challenging task. In this regard, carboxylate reductases (CARs) are promising catalysts provided by nature that are able to accomplish this task in just one step, avoiding over-reduction to the alcohol product. However, the heterologous expression of CARs can be quite difficult due to the excessive formation of insoluble protein, thus hindering further characterization and application of the enzyme. Here, the heterologous production of the carboxylate reductase from Nocardia otitidiscaviarum (NoCAR) was optimized by a combination of i) optimized cultivation conditions, ii) post-translational modification with a phosphopantetheinyl transferase and iii) selection of an appropriate expression strain. Especially, the selection of Escherichia coli tuner cells as host had a strong effect on the final 110-fold increase in the specific activity of NoCAR. This highly active NoCAR was used to reduce sodium benzoate to benzaldehyde, and it was successfully assembled with an in vitro regeneration of ATP and NADPH, being capable of reducing about 30 mM sodium benzoate with high selectivity in only 2 h of reaction.

Identification of a secreted superoxide dismutase (SOD) from Nocardia seriolae which induces apoptosis in fathead minnow (FHM) cells.

Fish nocardiosis is a chronic systemic granulomatous disease, and Nocardia seriolae is the main pathogen. The pathogenesis and virulence factors of N. seriolae are not fully understood. Secreted superoxide dismutase (SOD) may be a virulence factor found by a comparative bioinformatics analysis of the whole genome sequence of N. seriolae and the virulence factor database (VFDB). In order to determine the subcellular localization and study the preliminary function of SOD from N. seriolae (NsSOD), gene cloning, secreted protein identification, subcellular localization in fish cells, and apoptosis detection of NsSOD were carried out in this study. Subcellular localization research revealed that NsSOD-GFP fusion proteins were evenly distributed in the cytoplasm. Furthermore, apoptotic bodies were observed in the transfected FHM cells by the overexpression of protein NsSOD. Then, assays of mitochondrial membrane potential (ΔΨm) value, caspase-3 activity and apoptosis-related genes (Bax, Bid, Bad and Bcl-2) mRNA expression were conducted. The results showed that ΔΨm was decreased, and caspase-3 was significantly activated. The mRNA expression of the Bad gene showed significant up-regulated expression at 24 h.p.t., while Bid and Bax genes showed significant up-regulated expression at 72 and 96 h.p.t. and anti-apoptotic gene (Bcl-2) was down-regulated in NsSOD overexpressed cells. Taken together, the results indicated that the protein NsSOD might be involved in apoptosis regulation. This study may lay the foundations for further studies on the function of NsSOD and promote the understanding of the virulence factors and the pathogenic mechanisms of N. seriolae.

Inhibition Activity of Avibactam against Nocardia farcinica ß-Lactamase FARIFM10152.

Nocardia farcinica, one of the most frequent pathogenic species responsible for nocardiosis, is characterized by frequent brain involvement and resistance to ß-lactams mediated by a class A ß-lactamase. Kinetic parameters for hydrolysis of various ß-lactams by FARIFM10152 from strain IFM 10152 were determined by spectrophotometry revealing a high catalytic activity (kcat/Km ) for amoxicillin, aztreonam, and nitrocefin. For cephems, kcat/Km was lower but remained greater than 104 M-1 s-1 A low catalytic activity was observed for meropenem, imipenem, and ceftazidime hydrolysis. FARIFM10152 inhibition by avibactam and clavulanate was compared using nitrocefin as a reporter substrate. FARIFM10152 was efficaciously inhibited by avibactam with a carbamoylation rate constant (k2/Ki ) of (1.7 ± 0.3) × 104 M-1 s-1 The 50% effective concentrations (EC50s) of avibactam and clavulanate were 0.060 ± 0.007 µM and 0.28 ± 0.06 µM, respectively. Amoxicillin, cefotaxime, imipenem, and meropenem MICs were measured for ten clinical strains in the presence of avibactam and clavulanate. At 4 µg/ml, avibactam and clavulanate restored amoxicillin susceptibility in all but one of the tested strains but had no effect on the MICs of cefotaxime, imipenem, and meropenem. At 0.4 µg/ml, amoxicillin susceptibility (MIC ≤ 8 µg/ml) was restored for 9 out of 10 strains by avibactam but only for 4 out of 10 strains by clavulanate. Together, these results indicate that avibactam was at least as potent as clavulanate, suggesting that the amoxicillin-avibactam combination could be considered as an option for the rescue treatment of N. farcinica infections if clavulanate cannot be used.

Identification of a cell-wall peptidase (NlpC/P60) from Nocardia seriolae which induces apoptosis in fathead minnow cells.

Nocardia seriolae, a Gram-positive bacterium, is the main pathogen of fish nocardiosis. Protein NlpC/P60 is a cell-wall peptidase and a potential virulence factor of N. seriolae. Subcellular localization research revealed that both NlpC/P60-GFP and NlpC/P60Δsig-GFP fusion proteins were evenly distributed in the whole cell of fathead minnow (FHM) cells. Furthermore, typical apoptotic features, such as nuclear pyrosis and apoptotic bodies, were observed in the transfected FHM cells and grouper spleen cells by the overexpression of protein NlpC/P60. Then, quantitative assays of mitochondrial membrane potential (ΔΨm) value, caspase-3 activity and apoptosis-related gene (Bax, BNIP3, TNF1 and TNF6) mRNA expression were conducted. The results showed that ΔΨm was decreased, caspase-3 was significantly activated, and the mRNA expression of pro-apoptotic genes (Bax and BNIP3) and tumour necrosis factors (TNF1 and TNF6) was up-regulated in NlpC/P60-overexpressed cells. Taken together, the results indicated that the protein NlpC/P60 of N. seriolae might involve in apoptosis regulation. This study may lay the foundation for further study on the function of N. seriolae NlpC/P60 and promote the understanding of the virulence factors and pathogenic mechanism of N. seriolae.

Diterpene Biosynthesis in Actinomycetes: Studies on Cattleyene Synthase and Phomopsene Synthase.

Three diterpene synthases from actinomycetes have been studied. The first enzyme from Streptomyces cattleya produced the novel compound cattleyene. The other two enzymes from Nocardia testacea and Nocardia rhamnosiphila were identified as phomopsene synthases. The cyclisation mechanism of cattleyene synthase and the EIMS fragmentation mechanism of its product were extensively studied by incubation experiments with isotopically labelled precursors. Oxidative transformations expanded the chemical space of these unique diterpenes.

Structural Evidence for Rifampicin Monooxygenase Inactivating Rifampicin by Cleaving Its Ansa-Bridge.

Rifampicin monooxygenase (RIFMO) decreases the potency of rifampicin (RIF) by converting it to oxidative products. Further decomposition of RIF has been observed in bacteria producing RIFMO and contributes to RIFMO-mediated drug resistance. Here we report the first crystal structure of RIFMO in complex with the hydroxylated RIF product. The 2.10 Å resolution structure reveals a breach of the ansa aliphatic chain of RIF between naphthoquinone C2 and amide N1. Our data suggest that RIFMO catalyzes the hydroxylation of RIF at the C2 atom followed by cleavage of the ansa linkage, which leads to inactivation of the antibiotic by preventing key contacts with the RNA polymerase target.

Characterization of Carboxylic Acid Reductases for Biocatalytic Synthesis of Industrial Chemicals.

Carboxylic acid reductases (CARs) catalyze the reduction of a broad range of carboxylic acids into aldehydes, which can serve as common biosynthetic precursors to many industrial chemicals. This work presents the systematic biochemical characterization of five carboxylic acid reductases from different microorganisms, including two known and three new ones, by using a panel of short-chain dicarboxylic acids and hydroxy acids, which are common cellular metabolites. All enzymes displayed broad substrate specificities. Higher catalytic efficiencies were observed when the carbon chain length, either of the dicarboxylates or of the terminal hydroxy acids, was increased from C2 to C6 . In addition, when substrates of the same carbon chain length are compared, carboxylic acid reductases favor hydroxy acids over dicarboxylates as their substrates. Whole-cell bioconversions of eleven carboxylic acid substrates into the corresponding alcohols were investigated by coupling the CAR activity with that of an aldehyde reductase in Escherichia coli hosts. Alcohol products were obtained in yields ranging from 0.5 % to 71 %. The de novo stereospecific biosynthesis of propane-1,2-diol enantiomer was successfully demonstrated with use of CARs as the key pathway enzymes. E. coli strains accumulated 7.0 mm (R)-1,2-PDO (1.0 % yield) or 9.6 mm (S)-1,2-PDO (1.4 % yield) from glucose. This study consolidates carboxylic acid reductases as promising enzymes for sustainable synthesis of industrial chemicals.

The Structure of the Antibiotic Deactivating, N-hydroxylating Rifampicin Monooxygenase.

Rifampicin monooxygenase (RIFMO) catalyzes the N-hydroxylation of the natural product antibiotic rifampicin (RIF) to 2'-N-hydroxy-4-oxo-rifampicin, a metabolite with much lower antimicrobial activity. RIFMO shares moderate sequence similarity with well characterized flavoprotein monooxygenases, but the protein has not been isolated and characterized at the molecular level. Herein, we report crystal structures of RIFMO from Nocardia farcinica, the determination of the oligomeric state in solution with small angle x-ray scattering, and the spectrophotometric characterization of substrate binding. The structure identifies RIFMO as a class A flavoprotein monooxygenase and is similar in fold and quaternary structure to MtmOIV and OxyS, which are enzymes in the mithramycin and oxytetracycline biosynthetic pathways, respectively. RIFMO is distinguished from other class A flavoprotein monooxygenases by its unique middle domain, which is involved in binding RIF. Small angle x-ray scattering analysis shows that RIFMO dimerizes via the FAD-binding domain to form a bell-shaped homodimer in solution with a maximal dimension of 110 Å. RIF binding was monitored using absorbance at 525 nm to determine a dissociation constant of 13 µm Steady-state oxygen consumption assays show that NADPH efficiently reduces the FAD only when RIF is present, implying that RIF binds before NADPH in the catalytic scheme. The 1.8 Å resolution structure of RIFMO complexed with RIF represents the precatalytic conformation that occurs before formation of the ternary E-RIF-NADPH complex. The RIF naphthoquinone blocks access to the FAD N5 atom, implying that large conformational changes are required for NADPH to reduce the FAD. A model for these conformational changes is proposed.

De novo biosynthesis of trans-cinnamic acid derivatives in Saccharomyces cerevisiae.

The production of natural aroma compounds is an expanding field within the branch of white biotechnology. Three aromatic compounds of interest are cinnamaldehyde, the typical cinnamon aroma that has applications in agriculture and medical sciences, as well as cinnamyl alcohol and hydrocinnamyl alcohol, which have applications in the cosmetic industry. Current production methods, which rely on extraction from plant materials or chemical synthesis, are associated with drawbacks regarding scalability, production time, and environmental impact. These considerations make the development of a sustainable microbial-based production highly desirable. Through steps of rational metabolic engineering, we engineered the yeast Saccharomyces cerevisiae as a microbial host to produce trans-cinnamic acid derivatives cinnamaldehyde, cinnamyl alcohol, and hydrocinnamyl alcohol, from externally added trans-cinnamic acid or de novo from glucose as a carbon source. We show that the desired products can be de novo synthesized in S. cerevisiae via the heterologous overexpression of the genes encoding phenylalanine ammonia lyase 2 from Arabidopsis thaliana (AtPAL2), aryl carboxylic acid reductase (acar) from Nocardia sp., and phosphopantetheinyl transferase (entD) from Escherichia coli, together with endogenous alcohol dehydrogenases. This study provides a proof of concept and a strain that can be further optimized for production of high-value aromatic compounds.

An unprecedented NADPH domain conformation in lysine monooxygenase NbtG provides insights into uncoupling of oxygen consumption from substrate hydroxylation.

N-Hydroxylating monooxygenases are involved in the biosynthesis of iron-chelating hydroxamate-containing siderophores that play a role in microbial virulence. These flavoenzymes catalyze the NADPH- and oxygen-dependent hydroxylation of amines such as those found on the side chains of lysine and ornithine. In this work we report the biochemical and structural characterization of Nocardia farcinica Lys monooxygenase (NbtG), which has similar biochemical properties to mycobacterial homologs. NbtG is also active on d-Lys, although it binds l-Lys with a higher affinity. Differently from the ornithine monooxygenases PvdA, SidA, and KtzI, NbtG can use both NADH and NADPH and is highly uncoupled, producing more superoxide and hydrogen peroxide than hydroxylated Lys. The crystal structure of NbtG solved at 2.4 Å resolution revealed an unexpected protein conformation with a 30° rotation of the NAD(P)H domain with respect to the flavin adenine dinucleotide (FAD) domain that precludes binding of the nicotinamide cofactor. This "occluded" structure may explain the biochemical properties of NbtG, specifically with regard to the substantial uncoupling and limited stabilization of the C4a-hydroperoxyflavin intermediate. Biological implications of these findings are discussed.

De novo biosynthesis of Gastrodin in Escherichia coli.

Gastrodin, a phenolic glycoside, is the key ingredient of Gastrodia elata, a notable herbal plant that has been used to treat various conditions in oriental countries for centuries. Gastrodin is extensively used clinically for its sedative, hypnotic, anticonvulsive and neuroprotective properties in China. Gastrodin is usually produced by plant extraction or chemical synthesis, which has many disadvantages. Herein, we report unprecedented microbial synthesis of gastrodin via an artificial pathway. A Nocardia carboxylic acid reductase, endogenous alcohol dehydrogenases and a Rhodiola glycosyltransferase UGT73B6 transformed 4-hydroxybenzoic acid, an intermediate of ubiquinone biosynthesis, into gastrodin in Escherichia coli. Pathway genes were overexpressed to enhance metabolic flux toward precursor 4-hydroxybenzyl alcohol. Furthermore, the catalytic properties of the UGT73B6 toward phenolic alcohols were improved through directed evolution. The finally engineered strain produced 545mgl(-1) gastrodin in 48h. This work creates a new route to produce gastrodin, instead of plant extractions and chemical synthesis.

Identification of structural determinants of NAD(P)H selectivity and lysine binding in lysine N(6)-monooxygenase.

l-lysine (l-Lys) N(6)-monooxygenase (NbtG), from Nocardia farcinica, is a flavin-dependent enzyme that catalyzes the hydroxylation of l-Lys in the presence of oxygen and NAD(P)H in the biosynthetic pathway of the siderophore nocobactin. NbtG displays only a 3-fold preference for NADPH over NADH, different from well-characterized related enzymes, which are highly selective for NADPH. The structure of NbtG with bound NAD(P)(+) or l-Lys is currently not available. Herein, we present a mutagenesis study targeting M239, R301, and E216. These amino acids are conserved and located in either the NAD(P)H binding domain or the l-Lys binding pocket. M239R resulted in high production of hydrogen peroxide and little hydroxylation with no change in coenzyme selectivity. R301A caused a 300-fold decrease on kcat/Km value with NADPH but no change with NADH. E216Q increased the Km value for l-Lys by 30-fold with very little change on the kcat value or in the binding of NAD(P)H. These results suggest that R301 plays a major role in NADPH selectivity by interacting with the 2'-phosphate of the adenine-ribose moiety of NADPH, while E216 plays a role in l-Lys binding.

Characterizing and predicting carboxylic acid reductase activity for diversifying bioaldehyde production.

Chemicals with aldehyde moieties are useful in the synthesis of polymerization reagents, pharmaceuticals, pesticides, flavors, and fragrances because of their high reactivity. However, chemical synthesis of aldehydes from carboxylic acids has unfavorable thermodynamics and limited specificity. Enzymatically catalyzed reductive bioaldehyde synthesis is an attractive route that overcomes unfavorable thermodynamics by ATP hydrolysis in ambient, aqueous conditions. Carboxylic acid reductases (Cars) are particularly attractive, as only one enzyme is required. We sought to increase the knowledge base of permitted substrates for four Cars. Additionally, the Lys2 enzyme family was found to be mechanistically the same as Cars and two isozymes were also tested. Our results show that Cars prefer molecules where the carboxylic acid is the only polar/charged group. Using this data and other published data, we develop a support vector classifier (SVC) for predicting Car reactivity and make predictions on all carboxylic acid metabolites in iAF1260 and Model SEED.

Synthesis of chiral 2-alkanols from n-alkanes by a P. putida whole-cell biocatalyst.

The cytochrome P450 monooxygenase CYP154A8 from Nocardia farcinica was previously found to catalyze hydroxylation of linear alkanes (C7 -C9 ) with a high regio- and stereoselectivity. The objective of this study was to integrate CYP154A8 along with suitable redox partners into a whole-cell system for the production of chiral 2-alkanols starting from alkanes. Both recombinant Escherichia coli and Pseudomonas putida whole-cell biocatalysts tested for this purpose showed the ability to produce chiral alkanols, but a solvent tolerant P. putida strain demonstrated several advantages in the applied biphasic reaction system. The optimized P. putida whole-cell system produced ∼16 mM (S)-2-octanol with 87% ee from octane, which is more than sevenfold higher than the previously described system with isolated enzymes. The achieved enantiopurity of the product could further be increased up to 99% ee by adding an alcohol dehydrogenase (ADH) to the alkane-oxidizing P. putida whole-cell systems. By using this setup for the individual conversions of heptane, octane or nonane, 2.6 mM (S)-2-heptanol with 91% ee, 5.4 mM (S)-2-octanol with 97% ee, or 5.5 mM (S)-2-nonanol with 97% ee were produced, respectively. The achieved concentrations of chiral 2-alkanols are the highest reported for a P450-based whole-cell system so far. Biotechnol. Bioeng. 2016;113: 1845-1852. © 2016 Wiley Periodicals, Inc.

[GLUCOSE METABOLISM IN SURFACTANTS PRODUCER NOCARDIA VACCINII IMV B-7405].

Key enzymes of glucose metabolism were detected in the cells of surfactants producer Nocardia vaccinii IMV B-7405 grown on this substrate. It has been established that glucose catabolism is performed through gluconate (FAD(+)-dependent glucose dehydrogenase activity 698 ± 35 nmol x min(-1) x mg(-1) of protein). Oxidation of gluconate to 6-phosphogluconate is catalised by gluconokinase (178 ± 9 nmol x min(-1) x mg(-1) of protein). 6-Phosphogluconate was involved into pentose phosphate cycle by constitutive NADP(+)-dependent 6-phosphogluconate dehydrogenase (activity 357 ± 17 nmol x min(-1) x mg(-1) of protein). The data obtained serve as the basis for theoretical calculations of optimal molar ratio of concentrations of energetically nonequivalent substrates for intensifying the surfactants synthesis on their mixture.

Genome based analysis of type-I polyketide synthase and nonribosomal peptide synthetase gene clusters in seven strains of five representative Nocardia species.

BACKGROUND: Actinobacteria of the genus Nocardia usually live in soil or water and play saprophytic roles, but they also opportunistically infect the respiratory system, skin, and other organs of humans and animals. Primarily because of the clinical importance of the strains, some Nocardia genomes have been sequenced, and genome sequences have accumulated. Genome sizes of Nocardia strains are similar to those of Streptomyces strains, the producers of most antibiotics. In the present work, we compared secondary metabolite biosynthesis gene clusters of type-I polyketide synthase (PKS-I) and nonribosomal peptide synthetase (NRPS) among genomes of representative Nocardia species/strains based on domain organization and amino acid sequence homology. RESULTS: Draft genome sequences of Nocardia asteroides NBRC 15531(T), Nocardia otitidiscaviarum IFM 11049, Nocardia brasiliensis NBRC 14402(T), and N. brasiliensis IFM 10847 were read and compared with published complete genome sequences of Nocardia farcinica IFM 10152, Nocardia cyriacigeorgica GUH-2, and N. brasiliensis HUJEG-1. Genome sizes are as follows: N. farcinica, 6.0 Mb; N. cyriacigeorgica, 6.2 Mb; N. asteroides, 7.0 Mb; N. otitidiscaviarum, 7.8 Mb; and N. brasiliensis, 8.9 - 9.4 Mb. Predicted numbers of PKS-I, NRPS, and PKS-I/NRPS hybrid clusters ranged between 4-11, 7-13, and 1-6, respectively, depending on strains, and tended to increase with increasing genome size. Domain and module structures of representative or unique clusters are discussed in the text. CONCLUSION: We conclude the following: 1) genomes of Nocardia strains carry as many PKS-I and NRPS gene clusters as those of Streptomyces strains, 2) the number of PKS-I and NRPS gene clusters in Nocardia strains varies substantially depending on species, and N. brasiliensis strains carry the largest numbers of clusters among the species studied, 3) the seven Nocardia strains studied in the present work have seven common PKS-I and/or NRPS clusters, some of whose products are yet to be studied, and 4) different N. brasiliensis strains have some different gene clusters of PKS-I/NRPS, although the rest of the clusters are common within the N. brasiliensis strains. Genome sequencing suggested that Nocardia strains are highly promising resources in the search of novel secondary metabolites.

Enzyme-substrate complex structures of CYP154C5 shed light on its mode of highly selective steroid hydroxylation.

CYP154C5 from Nocardia farcinica is a bacterial cytochrome P450 monooxygenase active on steroid molecules. The enzyme has recently been shown to exhibit exclusive regioselectivity and stereoselectivity in the conversion of various pregnans and androstans, yielding 16α-hydroxylated steroid products. This makes the enzyme an attractive candidate for industrial application in steroid hormone synthesis. Here, crystal structures of CYP154C5 in complex with four different steroid molecules were solved at resolutions of up to 1.9 Å. These are the first reported P450 structures from the CYP154 family in complex with a substrate. The active site of CYP154C5 forms a flattened hydrophobic channel with two opposing polar regions, perfectly resembling the size and polarity distribution of the steroids and thus resulting in highly specific steroid binding with Kd values in the range 10-100 nM. Key enzyme-substrate interactions were identified that accounted for the exclusive regioselectivity and stereoselectivity of the enzyme. Additionally, comparison of the four CYP154C5-steroid structures revealed distinct structural differences, explaining the observed variations in kinetic data obtained for this P450 with the steroids pregnenolone, dehydroepiandrosterone, progesterone, androstenedione, testosterone and nandrolone. This will facilitate the generation of variants with improved activity or altered selectivity in the future by means of protein engineering.

Identification of fluorinases from Streptomyces sp MA37, Norcardia brasiliensis, and Actinoplanes sp N902-109 by genome mining.

The fluorinase is an enzyme that catalyses the combination of S-adenosyl-L-methionine (SAM) and a fluoride ion to generate 5'-fluorodeoxy adenosine (FDA) and L-methionine through a nucleophilic substitution reaction with a fluoride ion as the nucleophile. It is the only native fluorination enzyme that has been characterised. The fluorinase was isolated in 2002 from Streptomyces cattleya, and, to date, this has been the only source of the fluorinase enzyme. Herein, we report three new fluorinase isolates that have been identified by genome mining. The novel fluorinases from Streptomyces sp. MA37, Nocardia brasiliensis, and an Actinoplanes sp. have high homology (80-87 % identity) to the original S. cattleya enzyme. They all possess a characteristic 21-residue loop. The three newly identified genes were overexpressed in E. coli and shown to be fluorination enzymes. An X-ray crystallographic study of the Streptomyces sp. MA37 enzyme demonstrated that it is almost identical in structure to the original fluorinase. Culturing of the Streptomyces sp. MA37 strain demonstrated that it not only also elaborates the fluorometabolites, fluoroacetate and 4-fluorothreonine, similar to S. cattleya, but this strain also produces a range of unidentified fluorometabolites. These are the first new fluorinases to be reported since the first isolate, over a decade ago, and their identification extends the range of fluorination genes available for fluorination biotechnology.