Hasse B. Rasmussen

CYP83A1 and CYP83B1, Two Nonredundant Cytochrome P450 Enzymes Metabolizing Oximes in the Biosynthesis of Glucosinolates in Arabidopsis

In the glucosinolate pathway, the postoxime enzymes have been proposed to have low specificity for the side chain and high specificity for the functional group. Here, we provide biochemical evidence for the functional role of the two cytochromes P450, CYP83A1 and CYP83B1, from Arabidopsis in oxime metabolism in the biosynthesis of glucosinolates. In a detailed analysis of the substrate specificities of the recombinant enzymes heterologously expressed in yeast (Saccharomyces cerevisiae), we show that aliphatic oximes derived from chain-elongated homologs of methionine are efficiently metabolized by CYP83A1, whereas CYP83B1 metabolizes these substrates with very low efficiency. Aromatic oximes derived from phenylalanine, tryptophan, and tyrosine are metabolized by both enzymes, although CYP83B1 has higher affinity for these substrates than CYP83A1, particularly in the case of indole-3-acetaldoxime, where there is a 50-fold difference in Km value. The data show that CYP83A1 and CYP83B1 are nonredundant enzymes under physiologically normal conditions in the plant. The ability of CYP83A1 to metabolize aromatic oximes, albeit at small levels, explains the presence of indole glucosinolates at various levels in different developmental stages of the CYP83B1 knockout mutant, rnt1-1. Plants overexpressing CYP83B1 contain elevated levels of aliphatic glucosinolates derived from methionine homologs, whereas the level of indole glucosinolates is almost constant in the overexpressing lines. Together with the previous characterization of the members of the CYP79 family involved in oxime production, this work provides a framework for metabolic engineering of glucosinolates and for further dissection of the glucosinolate pathway.

Novel inhibitory activity of the Staphylococcus aureus NorA efflux pump by a kaempferol rhamnoside isolated from Persea lingue Nees

OBJECTIVES To isolate a plant-derived compound with efflux inhibitory activity towards the NorA transporter of Staphylococcus aureus. METHODS Bioassay-guided isolation was used, with inhibition of ethidium bromide efflux via NorA as a guide. Characterization of activity was carried out using MIC determination and potentiation studies of a fluoroquinolone antibiotic in combination with the isolated compound. Everted membrane vesicles of Escherichia coli cells enriched with NorA were prepared to study efflux inhibitory activity in an isolated manner. RESULTS The ethanolic extract of Persea lingue was subjected to bioassay-guided fractionation and led to the isolation of the known compound kaempferol-3-O-α-L-(2,4-bis-E-p-coumaroyl)rhamnoside (compound 1). Evaluation of the dose-response relationship of compound 1 showed that ethidium bromide efflux was inhibited, with an IC(50) value of 2 μM. The positive control, reserpine, was found to have an IC(50) value of 9 μM. Compound 1 also inhibited NorA in enriched everted membrane vesicles of E. coli. Potentiation studies revealed that compound 1 at 1.56 mg/L synergistically increased the antimicrobial activity of ciprofloxacin 8-fold against a NorA overexpresser, and the synergistic activity was exerted at a fourth of the concentration necessary for reserpine. Compound 1 was not found to exert a synergistic effect on ciprofloxacin against a norA deletion mutant. The 2,3-coumaroyl isomer of compound 1 has been shown previously not to cause acute toxicity in mice at 20 mg/kg/day. CONCLUSIONS Our results show that compound 1 acts through inhibition of the NorA efflux pump. Combination of compound 1 with subinhibitory concentrations of ciprofloxacin renders a wild-type more susceptible and a NorA overexpresser S. aureus susceptible.

MAO-A inhibitory activity of quercetin from Calluna vulgaris (L.) Hull.

AIM OF THE STUDY This study investigated MAO-A inhibitory activity of methanol extract of Calluna vulgaris (L.) Hull., which traditionally has been used as a nerve calming remedy. MATERIALS AND METHODS A methanolic extract of Calluna vulgaris was partitioned against heptane, ethyl acetate and water. The three fractions were tested in a photometric peroxidase linked MAO-A bioassay. The ethyl acetate phase showed the highest MAO-A inhibitory activity. Quercetin was isolated by VLC through bioassay-guided fractionation and purified by re-crystallisation. The structure was elucidated by LC-MS and (1)H NMR. RESULTS The IC(50)-value for MAO-A inhibition by quercetin was 18+/-0.2 microM in an assay where the IC(50)-value for MAO-A inhibition by clorgylin was 0.2+/-0.02 microM. CONCLUSION The content of quercetin in Calluna vulgaris might explain the reported nerve calming effect of the plant.

Amides from Piper capense with CNS Activity – A Preliminary SAR Analysis

Piper capense L.f. (Piperaceae) is used traditionally in South Africa as a sleep inducing remedy. Bioassay-guided fractionation of the roots of P. capense led to the isolation of piperine (1) and 4,5-dihydropiperine (2), which showed moderate affinity for the benzodiazepine site on the GABAA receptor (IC50 values of 1.2 mM and 1.0 mM, respectively). The present study suggests that strict structural properties of the amides are essential for affinity. Taken together, these observations suggest that the carbon chain must contain not less than four carbons, and that a conjugated double bond, adjacent to the amide group, is necessary for binding to the receptor and that the amine part should be bulky.

New bisbenzylisoquinoline alkaloid from Laureliopsis philippiana.

Phytochemical investigation of Laureliopsis philippiana resulted in isolation of a new bisbenzylisoquinoline alkaloid (1) named laureliopsine A. The structure was established by spectroscopic methods, including 2D homo- and heteronuclear NMR experiments. This finding of a bisbenzylisoquinoline alkaloid in Laureliopsis supports its close relationship to Atherosperma and its taxonomic segregation from Laurelia.

Carroll rearrangement to construct the norneolignan skeleton

A three-step synthesis of the norneolignan skeleton involving the Carroll rearrangement as the key step has been developed and used for synthesis of the norneolignan skeleton, 1,3-diphenylpenta-1,4-diene and hinokiresinol.

Cinnamamides from Piper capense with affinity to the benzodiazepine site on the GABAA receptor

Epilepsy is among the most prevalent of the serious neurological disorders, affecting from 0.5–1.0% of the worlds population and the prevalence of epilepsy in developing countries is generally higher than in developed countries [1]. Recently, plants used in South African traditional medicine for treatment of epilepsy and convulsion were examined for their pharmacological properties in the search for new and better anti-epileptic drugs [2, 3, 4]. The root of Piper capense L.f. (Piperaceae) is used in traditional South African medicine. A bioassay guided fractionation of an ethanolic extract using VLC, HPLC-UV, GC-MS and 1H-NMR led to the isolation and identification of cinnamamides with affinity to the benzodiazepine site on the GABAA receptor in the [3H]flumazenil receptor binding assay. The isolated cinnamides piperine and 4,5 dihydropiperine binded to the receptor with IC50 values of 0.9 and 1.7±mM respectively. . References: 1. Sander and Shorvon (1996)J Neurol Neurosurg Psych 65:433–443. 2. Svenningsen et al. (2006)J Ethnopharmacol. 103: 276–280. 3. Stafford, et al. (2005)J Ethnopharmacol 100: 210–215. 4. Risa, et al. (2004)J Ethnopharmacol 93:177–182