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

Enzymatic radiosynthesis of a 18F-Glu-Ureido-Lys ligand for the prostate-specific membrane antigen (PSMA)

We thank the Engineering and Physical Sciences Research Council, UK, for a research grant (EP/M01262X/1).Peer reviewedPostprin

An engineered E. coli strain for direct in vivo fluorination

This work was funded by the Industrial Biotechnology Innovation Centre (IBioIC) with support from GlaxoSmithKline, and also the EU Horizon 2020 (Sinfonia consortia).Selectively fluorinated compounds are found frequently in pharmaceutical and agrochemical products where currently 25–30 % of optimised compounds emerge from development containing at least one fluorine atom. There are many methods for the site‐specific introduction of fluorine, but all are chemical and they often use environmentally challenging reagents. Biochemical processes for C−F bond formation are attractive, but they are extremely rare. In this work, the fluorinase enzyme, originally identified from the actinomycete bacterium Streptomyces cattleya, is engineered into Escherichia coli in such a manner that the organism is able to produce 5′‐fluorodeoxyadenosine (5′‐FDA) from S‐adenosyl‐l‐methionine (SAM) and fluoride in live E. coli cells. Success required the introduction of a SAM transporter and deletion of the endogenous fluoride efflux capacity in order to generate an E. coli host that has the potential for future engineering of more elaborate fluorometabolites.PostprintPeer reviewe

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

Evaluation of serological cross-reactivity and cross-neutralization between the United States porcine epidemic diarrhea virus prototype and S-INDEL-variant strains

BACKGROUND: At least two genetically different porcine epidemic diarrhea virus (PEDV) strains have been identified in the United States (U.S. PEDV prototype and S-INDEL-variant strains). The current serological assays offered at veterinary diagnostic laboratories for detection of PEDV-specific antibody are based on the U.S. PEDV prototype strain. The objectives of this study were: 1) isolate the U.S. PEDV S-INDEL-variant strain in cell culture; 2) generate antisera against the U.S. PEDV prototype and S-INDEL-variant strains by experimentally infecting weaned pigs; 3) determine if the various PEDV serological assays could detect antibodies against the U.S. PEDV S-INDEL-variant strain and vice versa. RESULTS: A U.S. PEDV S-INDEL-variant strain was isolated in cell culture in this study. Three groups of PEDV-negative, 3-week-old pigs (five pigs per group) were inoculated orally with a U.S. PEDV prototype isolate (previously isolated in our lab), an S-INDEL-variant isolate or virus-negative culture medium. Serum samples collected at 0, 7, 14, 21 and 28 days post inoculation were evaluated by the following PEDV serological assays: 1) indirect fluorescent antibody (IFA) assays using the prototype and S-INDEL-variant strains as indicator viruses; 2) virus neutralization (VN) tests against the prototype and S-INDEL-variant viruses; 3) PEDV prototype strain whole virus based ELISA; 4) PEDV prototype strain S1-based ELISA; and 5) PEDV S-INDEL-variant strain S1-based ELISA. The positive antisera against the prototype strain reacted to and neutralized both prototype and S-INDEL-variant viruses, and the positive antisera against the S-INDEL-variant strain also reacted to and neutralized both prototype and S-INDEL-variant viruses, as examined by IFA antibody assays and VN tests. Antibodies against the two PEDV strains could be detected by all three ELISAs although detection rates varied to some degree. CONCLUSIONS: These data indicate that the antibodies against U.S. PEDV prototype and S-INDEL-variant strains cross-reacted and cross-neutralized both strains in vitro. The current serological assays based on U.S. PEDV prototype strain can detect antibodies against both U.S. PEDV strains

High Genetic Diversity and Fine-Scale Spatial Structure in the Marine Flagellate Oxyrrhis marina (Dinophyceae) Uncovered by Microsatellite Loci

Free-living marine protists are often assumed to be broadly distributed and genetically homogeneous on large spatial scales. However, an increasing application of highly polymorphic genetic markers (e.g., microsatellites) has provided evidence for high genetic diversity and population structuring on small spatial scales in many free-living protists. Here we characterise a panel of new microsatellite markers for the common marine flagellate Oxyrrhis marina. Nine microsatellite loci were used to assess genotypic diversity at two spatial scales by genotyping 200 isolates of O. marina from 6 broad geographic regions around Great Britain and Ireland; in one region, a single 2 km shore line was sampled intensively to assess fine-scale genetic diversity. Microsatellite loci resolved between 1–6 and 7–23 distinct alleles per region in the least and most variable loci respectively, with corresponding variation in expected heterozygosities (He) of 0.00–0.30 and 0.81–0.93. Across the dataset, genotypic diversity was high with 183 genotypes detected from 200 isolates. Bayesian analysis of population structure supported two model populations. One population was distributed across all sampled regions; the other was confined to the intensively sampled shore, and thus two distinct populations co-occurred at this site. Whilst model-based analysis inferred a single UK-wide population, pairwise regional FST values indicated weak to moderate population sub-division (0.01–0.12), but no clear correlation between spatial and genetic distance was evident. Data presented in this study highlight extensive genetic diversity for O. marina; however, it remains a substantial challenge to uncover the mechanisms that drive genetic diversity in free-living microorganisms

Identification of d -arabinan-degrading enzymes in mycobacteria

Bacterial cell growth and division require the coordinated action of enzymes that synthesize and degrade cell wall polymers. Here, we identify enzymes that cleave the D-arabinan core of arabinogalactan, an unusual component of the cell wall of Mycobacterium tuberculosis and other mycobacteria. We screened 14 human gut-derived Bacteroidetes for arabinogalactan-degrading activities and identified four families of glycoside hydrolases with activity against the D-arabinan or D-galactan components of arabinogalactan. Using one of these isolates with exo-D-galactofuranosidase activity, we generated enriched D-arabinan and used it to identify a strain of Dysgonomonas gadei as a D-arabinan degrader. This enabled the discovery of endo- and exo-acting enzymes that cleave D-arabinan, including members of the DUF2961 family (GH172) and a family of glycoside hydrolases (DUF4185/GH183) that display endo-D-arabinofuranase activity and are conserved in mycobacteria and other microbes. Mycobacterial genomes encode two conserved endo-D-arabinanases with different preferences for the D-arabinan-containing cell wall components arabinogalactan and lipoarabinomannan, suggesting they are important for cell wall modification and/or degradation. The discovery of these enzymes will support future studies into the structure and function of the mycobacterial cell wall

Caspase 8 and maspin are downregulated in breast cancer cells due to CpG site promoter methylation

<p>Abstract</p> <p>Background</p> <p>Epigenetic changes associated with promoter DNA methylation results in silencing of several tumor suppressor genes that lead to increased risk for tumor formation and for progression of the cancer.</p> <p>Methods</p> <p>Methylation specific PCR (MSP) and bisulfite sequencing were used for determination of proapoptotic gene Caspase 8 (CASP8) and the tumor suppressor gene maspin promoter methylation in four breast cancer and two non-tumorigenic breast cell lines. Involvement of histone H3 methylation in those cell lines were examined by CHIP assay.</p> <p>Results</p> <p>The CpG sites in the promoter region of CASP8 and maspin were methylated in all four breast cancer cell lines but not in two non-tumorigenic breast cell lines. Demethylation agent 5-aza-2'-deoxycytidine (5-aza-dc) selectively inhibits DNA methyltransferases, DNMT3a and DNMT3b, and restored CASP8 and maspin gene expression in breast cancer cells. 5-aza-dc also reduced histone H3k9me2 occupancy on CASP8 promoter in SKBR3cells, but not in MCF-7 cells. Combination of histone deacetylase inhibitor Trichostatin A (TSA) and 5-aza-dc significant decrease in nuclear expression of Di-methyl histone H3-Lys27 and slight increase in acetyl histone H3-Lys9 in MCF-7 cells. CASP8 mRNA and protein level in MCF-7 cells were increased by the 5-aza-dc in combination with TSA. Data from our study also demonstrated that treatment with 5-FU caused a significant increase in unmethylated CASP8 and in CASP8 mRNA in all 3 cancer lines.</p> <p>Conclusions</p> <p>CASP8 and maspin expression were reduced in breast cancer cells due to promoter methylation. Selective application of demethylating agents could offer novel therapeutic opportunities in breast cancer.</p

Complete Genome Sequence of Mycoplasma suis and Insights into Its Biology and Adaption to an Erythrocyte Niche

Mycoplasma suis, the causative agent of porcine infectious anemia, has never been cultured in vitro and mechanisms by which it causes disease are poorly understood. Thus, the objective herein was to use whole genome sequencing and analysis of M. suis to define pathogenicity mechanisms and biochemical pathways. M. suis was harvested from the blood of an experimentally infected pig. Following DNA extraction and construction of a paired end library, whole-genome sequencing was performed using GS-FLX (454) and Titanium chemistry. Reads on paired-end constructs were assembled using GS De Novo Assembler and gaps closed by primer walking; assembly was validated by PFGE. Glimmer and Manatee Annotation Engine were used to predict and annotate protein-coding sequences (CDS). The M. suis genome consists of a single, 742,431 bp chromosome with low G+C content of 31.1%. A total of 844 CDS, 3 single copies, unlinked rRNA genes and 32 tRNAs were identified. Gene homologies and GC skew graph show that M. suis has a typical Mollicutes oriC. The predicted metabolic pathway is concise, showing evidence of adaptation to blood environment. M. suis is a glycolytic species, obtaining energy through sugars fermentation and ATP-synthase. The pentose-phosphate pathway, metabolism of cofactors and vitamins, pyruvate dehydrogenase and NAD+ kinase are missing. Thus, ribose, NADH, NADPH and coenzyme A are possibly essential for its growth. M. suis can generate purines from hypoxanthine, which is secreted by RBCs, and cytidine nucleotides from uracil. Toxins orthologs were not identified. We suggest that M. suis may cause disease by scavenging and competing for host' nutrients, leading to decreased life-span of RBCs. In summary, genome analysis shows that M. suis is dependent on host cell metabolism and this characteristic is likely to be linked to its pathogenicity. The prediction of essential nutrients will aid the development of in vitro cultivation systems

The Complete Genome Sequence of Fibrobacter succinogenes S85 Reveals a Cellulolytic and Metabolic Specialist

Fibrobacter succinogenes is an important member of the rumen microbial community that converts plant biomass into nutrients usable by its host. This bacterium, which is also one of only two cultivated species in its phylum, is an efficient and prolific degrader of cellulose. Specifically, it has a particularly high activity against crystalline cellulose that requires close physical contact with this substrate. However, unlike other known cellulolytic microbes, it does not degrade cellulose using a cellulosome or by producing high extracellular titers of cellulase enzymes. To better understand the biology of F. succinogenes, we sequenced the genome of the type strain S85 to completion. A total of 3,085 open reading frames were predicted from its 3.84 Mbp genome. Analysis of sequences predicted to encode for carbohydrate-degrading enzymes revealed an unusually high number of genes that were classified into 49 different families of glycoside hydrolases, carbohydrate binding modules (CBMs), carbohydrate esterases, and polysaccharide lyases. Of the 31 identified cellulases, none contain CBMs in families 1, 2, and 3, typically associated with crystalline cellulose degradation. Polysaccharide hydrolysis and utilization assays showed that F. succinogenes was able to hydrolyze a number of polysaccharides, but could only utilize the hydrolytic products of cellulose. This suggests that F. succinogenes uses its array of hemicellulose-degrading enzymes to remove hemicelluloses to gain access to cellulose. This is reflected in its genome, as F. succinogenes lacks many of the genes necessary to transport and metabolize the hydrolytic products of non-cellulose polysaccharides. The F. succinogenes genome reveals a bacterium that specializes in cellulose as its sole energy source, and provides insight into a novel strategy for cellulose degradation