Straightforward Regeneration of Reduced Flavin Adenine Dinucleotide Required for Enzymatic Tryptophan Halogenation

Flavin-dependent halogenases are known to regioselectively introduce halide substituents into aromatic moieties, for example, the indole ring of tryptophan. The process requires halide salts and oxygen instead of molecular halogen in the chemical halogenation. However, the reduced cofactor flavin adenine dinucleotide (FADH2) has to be regenerated using a flavin reductase. Consequently, coupled biocatalytic steps are usually applied for cofactor regeneration. Nicotinamide adenine dinucleotide (NADH) mimics can be employed stoichiometrically to replace enzymatic cofactor regeneration in biocatalytic halogenation. Chlorination of l-tryptophan is successfully performed using such NADH mimics. The efficiency of this approach has been compared to the previously established enzymatic regeneration system using the two auxiliary enzymes flavin reductase (PrnF) and alcohol dehydrogenase (ADH). The reaction rates of some of the tested mimics were found to exceed that of the enzymatic system. Continuous enzymatic halogenation reaction for reaction scale-up is also possible

X-ray, structural assignment and molecular docking study of dihydrogeodin from <i>Aspergillus Terreus</i> TM8

<p>A re-cultivation of the thermophilic fungus <i>Aspergillus terreus</i> TM8, and working up of its extract afforded the dichloro-benzophenone derivative, dihydrogeodin (<b>1</b>) in addition to the butyrolactones I (<b>2</b>), V (<b>3</b>) and VI (<b>4</b>). A literature surveying revealed one recent structural assignment trial for dihydrogeodin (<b>1</b>), however, with some inaccuracies. We report herein a full assignment of dihydrogeodin (<b>1</b>) using extensive study of 1D, 2D NMR and ESI HR mass data. For the first time as well, we report the planar structure of <b>1</b> using X-ray crystallography. Docking and molecular dynamic simulation of dihydrogeodin (<b>1</b>) on the isomerase cyclophilin A has revealed its significant potential activity as an antiviral and immunosuppressive agent.</p

Structure of apo BrvH compared to RebH bound to FAD, Trp and Cl^-.

<p><b>A:</b> The structural alignment was made in PyMOL based on the conserved “box” domain of the two halogenases (BrvH: 6–99, 162–416, 494–502 vs. RebH: 2–98, 167–426, 519–528; PDB ID 2oa1). FAD, Trp (both dark grey) and Cl^- (green) are present only in the structure of RebH. BrvH (blue) and RebH (grey) are structurally very similar. A major difference is found in the substrate binding site, which is accessible to the solvent in BrvH, while it is covered by a longer loop in RebH. <b>B:</b> Empty substrate binding site of BrvH overlaid with the tryptophan binding site of RebH containing Trp (dark grey). Several of the RebH residues directly contacting the bound Trp are shown as grey sticks along with the corresponding residues of BrvH shown in blue. The loops that are shown correspond to those in A. While the residues contacting the indole ring are conserved, those residues that form hydrogen bonds to the carboxylate and the α-amino group of Trp in RebH are not present in BrvH. K83 and E349 of BrvH correspond to the amino acids shown to be important for the catalytic mechanism in other halogenases (K79 and E357 in RebH).</p

Program for the site-directed mutagenesis PCR.

<p>Program for the site-directed mutagenesis PCR.</p

Phylogenetic tree of positive detected FHals from the Botany Bay metagenome in comparison to known FHals on amino acid level.

<p>The tree was constructed using neighbour joining method, bootstrap 1000, with the alignment based on amino acids by MEGA7 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0196797#pone.0196797.ref043" target="_blank">43</a>] and MUSCLE [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0196797#pone.0196797.ref039" target="_blank">39</a>]. 1–42: positive FHal hits from the Botany Bay metagenome; PyrH, PrnA, RebH, Thal: Tryptophan halogenases belonging to variant A and accept free substrate; PltA, CrpH, BhaA, CndH: FHals belonging to variant B and require carrier bound substrate. The amino acid sequences are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0196797#pone.0196797.s017" target="_blank">S3 Table</a>.</p

Gene cluster in the vicinity of gene 12 (K) in the Botany Bay metagenome.

<p>Upstream to the gene 12 (K), another FHal, 13 (L) was identified.</p

Isoshamixanthone: a new pyrano xanthone from endophytic <i>Aspergillus</i> sp. ASCLA and absolute configuration of epiisoshamixanthone

Isoshamixanthone (1), a new stereoisomeric pyrano xanthone together with the previously known fungal metabolites, epiisoshamixanthone (2), sterigmatocystin (3), arugosin C (4), norlichexanthone (5), diorcinol (6), ergosterol and methyllinoleate, were obtained from the endophytic fungal strain Aspergillus sp. ASCLA isolated from leaf tissues of the medicinal plant Callistemon subulatus. The chemical structure of the new xanthone (1) was elucidated by extensive 1D, 2D NMR, and ESI HR mass measurements, and by comparison with literature data. The constitutions and absolute configurations of 1 and epiisoshamixanthone (2) were additionally confirmed by X-ray crystallography. Compounds 1,2 were evaluated for their potential anticancer activity using the human cervix carcinoma cell line (KB-3-1). The antimicrobial activities of the fungal extract and compounds 1,2 were studied using a panel of pathogenic microorganisms as well. </p

Time course of the conversion of 0.05 mM indole by BrvH with NaBr over 25 minutes at 25 °C.

<p>The conversion rate was identified via RP-HPLC by determining the ratio of the peak areas of indole and bromoindole. The specific activity was defined between 0 and 15 minutes reaction time.</p

Antioxidant and cytotoxicity activities of δ-tocotrienol from the seeds of <i>Allophylus africanus</i>

Chemical investigation of Allophylus africanus P. Beauv fruits led to the isolation of a new δ-tocotrienol, 3α-hydroxy-δ-tocotrienol (1) together with eight known compounds (2-9). Compound (1) was allylated (1a) and prenylated (1 b and 1c) to give three new semi-synthesized derivatives which were fully characterized as: 6-O-allyl-3α-hydroxy-δ-tocotrienol (1a), 6-O-prenyl-3α-hydroxy-δ-tocotrienol (1 b) and 6-O,5-C-diprenyl-3α-hydroxy-δ-tocotrienol (1c). The structures of compounds were established using comprehensive spectroscopic analysis including UV, MS, 1 D NMR, 2 D NMR and by comparison with the corresponding literature data. Compound (1) and its semi-synthetic derivatives (1a-c) were tested for their antioxydant activity using DPPH radical scavenging assay and also for their cytotoxicity using human cervix carcinoma KB-3-1 cell lines. The results showed that compound (1) exhibited antioxidant activity with an IC50 value of 0.25 μM compared to the reference control trolox (26 µM); and good cytotoxic activity with IC50 values of 97 μM compared to the reference (+)-griseofulvin (IC50 between17-21 μM).</p

Comparison of the halide binding site in RebH and BrvH. FAD (dark grey) and Cl^- (green) are present only in the structure of RebH.

<p>Coordination of the halide takes place via the amide nitrogens of the backbone. Whilst the halide binding motif in RebH (grey) is T359, G360 in BrvH (blue) we see a T351, S352 at the same site. However, the amino acid exchange does not seem to alter the overall structure of the halide binding site, thus not giving a definite hint as to why BrvH would preferentially brominate.</p