Browse
Submit Data
Databases
API
Help

Dataset Information

0 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Isolation, expression and biochemical characterization of recombinant hyoscyamine-6?-hydroxylase from Brugmansia sanguinea - tuning the scopolamine production.

ABSTRACT: Hyoscyamine-6?-hydroxylase (H6H, EC 1.14.11.11) is a plant enzyme that catalyses the last two steps in the biosynthesis of the anticholinergic drug scopolamine, i.e. the hydroxylation of hyoscyamine to 6?-hydroxyhyoscyamine (anisodamine) and subsequent oxidative ring-closure to the 6,7-?-epoxide. A H6H gene homologue was isolated from the plant Brugmansia sanguinea (BsH6H) and recombinantly cloned into Escherichia coli, expressed and purified using an effective SUMO-fusion procedure. Enzymatic activity is approximately 40-fold higher for the first reaction step and the substrate affinity is comparable to other characterized H6H homologues (Km ? 60 ?M). Truncation of an H6H enzyme flexible N-terminal region yields an active and stable yet more compact enzyme version.

SUBMITTER: Fischer C

PROVIDER: S-EPMC6071785 | biostudies-literature | 2018 May

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Publications

Isolation, expression and biochemical characterization of recombinant hyoscyamine-6β-hydroxylase from Brugmansia sanguinea - tuning the scopolamine production.

Fischer Conrad C Kwon Moonhyuk M Ro Dae-Kun DK van Belkum Marco J MJ Vederas John C JC

MedChemComm 20180502 5

Hyoscyamine-6β-hydroxylase (H6H, EC 1.14.11.11) is a plant enzyme that catalyses the last two steps in the biosynthesis of the anticholinergic drug scopolamine, i.e. the hydroxylation of hyoscyamine to 6β-hydroxyhyoscyamine (anisodamine) and subsequent oxidative ring-closure to the 6,7-β-epoxide. A H6H gene homologue was isolated from the plant Brugmansia sanguinea (BsH6H) and recombinantly cloned into Escherichia coli, expressed and purified using an effective ...[more]

PMID: 30108978

Similar Datasets

Substrate Conformation Correlates with the Outcome of Hyoscyamine 6β-Hydroxylase Catalyzed Oxidation Reactions.

Project description:Hyoscyamine 6β-hydroxylase (H6H) is an α-ketoglutarate dependent mononuclear nonheme iron enzyme that catalyzes C6-hydroxylation of hyoscyamine and oxidative cyclization of the resulting product to give the oxirane natural product scopolamine. Herein, the chemistry of H6H is investigated using hyoscyamine derivatives with modifications at the C6 or C7 position as well as substrate analogues possessing a 9-azabicyclo[3.3.1]nonane core. Results indicate that hydroxyl rebound is unlikely to take place during the cyclization reaction and that the hydroxylase versus oxidative cyclase activity of H6H is correlated with the presence of an exo-hydroxy group having syn-periplanar geometry with respect to the adjacent H atom to be abstracted.

| S-EPMC6488034 | biostudies-literature

Expression of Brugmansia candida Hyoscyamine 6beta-Hydroxylase gene in Saccharomyces cerevisiae and its potential use as biocatalyst.

Project description:BACKGROUND: Tropane alkaloids, mainly hyoscyamine and scopolamine, are widely used in medicine due to their anticholinergic activity. Scopolamine has a higher demand being the more valuable alkaloid due to its fewer side effects and higher physiological activity. Anisodamine (6beta-hydroxyhyoscyamine) is the intermediate in the conversion of hyoscyamine into scopolamine. Current studies report that this alkaloid is potentially applicable in medicine. The gene that codifies for Hyoscyamine 6-beta hydroxylase, the enzyme responsible for hyoscyamine hydroxylation and epoxidation, leading to scopolamine was isolated from Brugmansia candida. RESULTS: The h6hcDNA was cloned into pYES2.1 and pYES2.1/V5-His-TOPO vectors to produce an untagged and a tagged protein, respectively. The H6H enzyme was produced in Saccharomyces cerevisiae in order to obtain a biological catalyst for potential industrial applications. Protein extracts of the induced yeast were analyzed by Western blot. The expression was detected 4 h after induction and no degradation was observed during the period assayed. The tagged and the untagged proteins were able to transform hyoscyamine, showing a functional expression of the h6hcDNA. CONCLUSION: The strains obtained in this work are promising and potentially applicable in biocatalytic processes.

| S-EPMC2430943 | biostudies-literature

Changes in Regioselectivity of H Atom Abstraction during the Hydroxylation and Cyclization Reactions Catalyzed by Hyoscyamine 6β-Hydroxylase.

Project description:Hyoscyamine 6β-hydroxylase (H6H) is an αKG-dependent nonheme iron oxidase that catalyzes the oxidation of hyoscyamine to scopolamine via two separate reactions: hydroxylation followed by oxidative cyclization. Both of these reactions are expected to involve H atom abstraction from each of two adjacent carbon centers (C6 vs C7) in the substrate. During hydroxylation, there is a roughly 85:1 preference for H atom abstraction from C6 versus C7; however, this inverts to a 1:16 preference during cyclization. Furthermore, 18O incorporation experiments in the presence of deuterated substrate are consistent with the catalytic iron(IV)-oxo complex being able to support the coordination of an additional ligand during hydroxylation. These observations suggest that subtle differences in the substrate binding configuration can have significant consequences for the catalytic cycle of H6H.

| S-EPMC6488026 | biostudies-literature

Regioselectivity of hyoscyamine 6β-hydroxylase-catalysed hydroxylation as revealed by high-resolution structural information and QM/MM calculations.

Project description:Hyoscyamine 6β-hydroxylase (H6H) is a bifunctional non-heme 2-oxoglutarate/Fe2+-dependent dioxygenase that catalyzes the two final steps in the biosynthesis of scopolamine. Based on high resolution crystal structures of H6H from Datura metel, detailed information on substrate binding was obtained that provided insights into the onset of the enzymatic process. In particular, the role of two prominent residues was revealed - Glu-116 that interacts with the tertiary amine located on the hyoscyamine tropane moiety and Tyr-326 that forms CH-π hydrogen bonds with the hyoscyamine phenyl ring. The structures were used as the basis for QM/MM calculations that provided an explanation for the regioselectivity of the hydroxylation reaction on the hyoscyamine tropane moiety (C6 vs. C7) and quantified contributions of active site residues to respective barrier heights.

| S-EPMC9705211 | biostudies-literature

Promoting the accumulation of scopolamine and hyoscyamine in Hyoscyamus niger L. through EMS based mutagenesis.

Project description:The overexploitation of medicinal plants is depleting gene pool at an alarming rate. In this scenario inducing the genetic variability through targeted mutations could be beneficial in generating varieties with increased content of active compounds. The present study aimed to develop a reproducible protocol for in vitro multiplication and mutagenesis of Hyoscyamus niger targeting putrescine N-methyltransferase (PMT) and 6β-hydroxy hyoscyamine (H6H) genes of alkaloid biosynthetic pathway. In vitro raised callus were treated with different concentrations (0.01% - 0.1%) of Ethyl Methane Sulfonate (EMS). Emerging multiple shoots and roots were obtained on the MS media supplemented with cytokinins and auxins. Significant effects on morphological characteristics were observed following exposure to different concentrations of EMS. EMS at a concentration of 0.03% was seen to be effective in enhancing the average shoot and root number from 14.5±0.30 to 22.2 ±0.77 and 7.2±0.12 to 8.8±0.72, respectively. The lethal dose (LD50) dose was calculated at 0.08% EMS. The results depicted that EMS has an intense effect on PMT and H6H gene expression and metabolite accumulation. The transcripts of PMT and H6H were significantly upregulated at 0.03-0.05% EMS compared to control. EMS treated explants showed increased accumulation of scopolamine (0.639 μg/g) and hyoscyamine (0.0344μg/g) compared to untreated.

| S-EPMC7241962 | biostudies-literature

Isolation and Biochemical Characterization of Recombinant Transketolase from Mycobacterium tuberculosis.

Project description:Transketolase, an enzyme of the pentose phosphate pathway, plays an important role in the functioning of mycobacteria. Using plasmid pET-19b carrying the Rv1449c gene of transketolase from Mycobacterium tuberculosis and an additional histidine tag, we isolated and purified recombinant transketolase and determined the conditions for obtaining the apoform of the protein. The Michaelis constants were evaluated for the thiamine diphosphate cofactor in the presence of magnesium and calcium ions. We found that the affinity of mycobacterial transketolase for thiamine diphosphate is by three orders of magnitude lower than that of the human enzyme. Analysis of the structural organization of the active centers of homologous enzymes showed that this difference is due to a replacement of lysine residues by less polar amino acid residues.

| S-EPMC9307985 | biostudies-literature

Isolation and characterization of microsatellite loci for Cornus sanguinea (Cornaceae).

Project description:To facilitate genetic and conservation research of Cornus sanguinea, microsatellite loci were isolated and 29 individuals from 11 German populations were genotyped. • Sixteen microsatellite loci were characterized from an enriched small insert genomic library. The number of alleles detected ranged from five to 11 per locus, observed heterozygosity ranged from 0.00 to 1.00, expected heterozygosity ranged from 0.65 to 0.90, and polymorphic information content ranged from 0.59 to 0.88. • The markers described in the study will allow further investigation of population dynamics and the degree of clonal reproduction within populations of C. sanguinea.

| S-EPMC4103144 | biostudies-literature

Enhanced production of hyoscyamine and scopolamine from genetically transformed root culture of Hyoscyamus reticulatus L. elicited by iron oxide nanoparticles.

Project description:The medicinal plant Hyoscyamus reticulatus L. is a rich source of hyoscyamine and scopolamine, the tropane alkaloids. The use of hairy root cultures has focused significant attention on production of important metabolites such as stable tropane alkaloid production. Elicitation is an effective approach to induce secondary metabolite biosynthetic pathways. Hairy roots were derived from cotyledon explants inoculated with Agrobacterium rhizogenes and elicited by iron oxide nanoparticles (FeNPs) at different concentrations (0, 450, 900, 1800, and 3600 mg L-1) for different exposure times (24, 48, and 72 h). The highest hairy root fresh and dry weights were found in the medium supplemented with 900 mg L-1 FeNPs. Antioxidant enzyme activity was significantly increased in induced hairy roots compared to non-transgenic roots. The highest hyoscyamine and scopolamine production (about fivefold increase over the control) was achieved with 900 and 450 mg L-1 FeNPs at 24 and 48 h of exposure time, respectively. This is the first report of the effect of FeNP elicitor on hairy root cultures of a medicinal plant. We suggest that FeNPs could be an effective elicitor in hairy root cultures in order to increase tropane alkaloid production.

| S-EPMC5423962 | biostudies-literature

Brugmansia arborea

Project description:Brugmansia arborea Genome sequencing and assembly

| PRJNA765960 | ENA

Tuning the transcriptional response to hypoxia through HIF prolyl- and asparaginyl-hydroxylase inhibition:Sequencing Data

Project description:The hypoxia inducible factor (HIF) system orchestrates cellular responses to hypoxia in animals. HIF is an /-heterodimeric transcription factor that regulates the expression of hundreds of genes in a context dependent manner. A hypoxia-sensing component of the HIF system involves oxygen-dependent catalysis by the HIF hydroxylases; in humans there are three HIF prolyl hydroxylases (PHD1-3) and an asparaginyl hydroxylase (FIH). PHD catalysis regulates HIF levels and FIH catalysis regulates HIF activity. How differences in HIF hydroxylation status relate to variations in the induction of HIF target gene transcription is unknown. We report studies using small molecule inhibitors of the HIF hydroxylases to investigate the extent to which HIF target gene upregulation is induced by reduced PHD catalysis. The results reveal substantial differences in the role of prolyl- and asparaginyl-hydroxylation in regulating hypoxia responsive genes in cells. Selective PHD inhibitors with different structural scaffolds behave similarly. However, under the tested conditions, a broad-spectrum 2OG dioxygenase inhibitor is a better mimic of the transcriptional response to hypoxia than the selective PHD inhibitors, consistent with an important role for FIH in the hypoxic transcriptional response. Indeed, combined application of selective PHD and FIH inhibitors resulted in transcriptional induction of a subset of genes that were not fully responsive to PHD inhibition alone. Thus, for the therapeutic regulation of HIF target genes, it is important to consider both PHD and FIH activity, and in the case of some sets of target genes, simultaneous inhibition of the PHDs and FIH catalysis may be preferable.

2016-10-25 | E-MTAB-4264 | biostudies-arrayexpress