Berberine (BBR) is a protoberberine alkaloid extracted from plants such as Coptis japonica (Ranunculaceae). In a previous report, we demonstrated the existence of a 11-hydroxylation pathway employed by BBR-utilizing bacteria for metabolism of BBR. In the present study, we report the identification of the genes brhA, brhB, and brhC as encoding a multicomponent BBR 11-hydroxylase in Burkholderia sp. strain CJ1. BrhA is belonging to the Rieske non-heme iron oxygenase (RO) family, a class of enzymes known to catalyze the first step in bacterial aromatic-ring hydroxylation. We further demonstrate that BrhA activity requires BrhB (ferredoxin reductase) and BrhC (ferredoxin) as electron transport chain components. A BLAST search revealed that BrhA exhibits 38% and 33% sequence identity to dicamba O-demethylase (DdmC; AY786443) and chloroacetanilide herbicides N-dealkylase (CndA; KJ461679), respectively. To our knowledge, this work represents the first report of a bacterial oxygenase catalyzing the metabolism of a polycyclic aromatic-ring alkaloid. Abbreviations: BBR: berberine; D-BBR: demethyleneberberine; H-BBR: 11-hydroxyberberine; HD-BBR: 11-hydroxydemethyleneberberine; HDBA: 2-hydroxy-3,4-dimethoxybenzeneacetic acid; PAL: palmatine; H-PAL: 11-hydroxypalmatine; BRU: berberrubine; Fd: ferredoxin; FdR: ferredoxin reductase; ETC: electron transport chain
Burkholderia sp. strain CJ1 was newly isolated as berberine (BBR) degrading bacteria from rhizosphere of Coptis japonica. CJ1 had the ability to utilize BBR as the sole carbon source and revealed that BBR metabolism via 11-hydroxylation and demethylenation pathway. It was also revealed that the 11-hydroxylation ability of BBR and palmatine (PAL) has induced by BBR.
Plants produce many specific secondary metabolites as a response to environmental stress, especially biological stress. These compounds show strong biological activities and high stability against degradation by microbes and animals. Berberine, a benzylisoquinoline alkaloid, is found in many plant species and has strong antimicrobial activity, and is often included in traditional herbal medicines. We previously investigated how berberine is degraded in nature and we isolated two berberine-utilizing bacteria. In this study, we characterized the gene encoding the enzyme that degrades the 2,3-methylenedioxy ring of berberine; this ring is important for its activity and stability. Further characterization of several other berberine-utilizing bacteria and the genes encoding key demethylenation enzymes revealed that these enzymes are tetrahydrofolate dependent and similar to demethylation enzymes such as GcvT. Because the degradation of O-methyl groups or the methylenedioxy ring in phenolic compounds such as lignin, lignan and many other natural products, including berberine, is the key step for the catabolism of these compounds, our discovery reveals the common origin of the catabolism of these stable chemicals in bacteria.Plants produce many secondary metabolites to respond to environmental stress, especially biological stress. These compounds have strong biological activities and high stability against degradation by microbes and animals. Berberine (BBR) is a benzylisoquinoline alkaloid that is produced by various higher plants, such as Coptis japonica, Berberis fremontii and Mahonia aquifolium 1 , and shows strong antimicrobial and chemical defense activities 2 . BBR was also used as an antidiarrheal in ancient times, and interest in its use toward improving intestinal microflora is increasing 3, 4 . Although few studies have characterized the structure-activity relationship of protoberberine alkaloids, chemicals with a methylenedioxy ring, such as BBR, tend to demonstrate more activity than those with dimethoxy or hydroxy groups, such as palmatine 5,6 .The metabolism of BBR has been mainly investigated in mammals. CYP2D6, CYP1A2 and CYP3A4 are responsible for metabolizing BBR to demethyleneberberine (D-BBR) in human liver microsomes 7 . Interestingly, BBR was not metabolized by the intestinal bacteria of humans and rats in anaerobic conditions 8,9 . Liquid chromatography/time-of-flight mass spectrometry analysis of bile, plasma and urine in rats revealed the presence of BBR metabolites, including thalifendine, berberrubine, D-BBR, jatrorrhizine, palmatine, columbamine, 3,9-demethyl-palmatine, hydroxylated BBR and hydroxylated D-BBR; BBR was converted into sulfate and glucuronic acid conjugates for excretion 10 .There is currently a new opportunity to study the environmental fate of BBR: two bacteria, Sphingobium sp. strain BD3100 and Rhodococcus sp. strain BD7100, that utilize BBR as their sole carbon source were isolated from the sludge of a berberine-producing factory 11 . The formation of demethyleneberberine ...
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