Identification of a multi-component berberine 11-hydroxylase from Burkholderia sp. strain CJ1

ABSTRACT

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     

Coassemble dopamine and GHK tripeptide into fluorescent nanoparticles for pH sensing

Fluorescent nanostructures have been widely applied to biomedical researches and clinical diagnosis such as biolabeling/imaging/sensing and have even acted as therapy reagents. Peptide‐based fluorescent nanostructures attract recent interest from biomedical researchers. Inspired by the natural existence of GHK‐Cu complex with a growth factor‐like effect in human blood, here we have developed a novel approach for designing nanosensors through the co‐assembling of two kinds of biomolecules. By making best use of both π‐π stacking between carbon rings and the easy‐oxidation property of an important transmitter molecule, dopamine (DA), we successfully built up a supersensitive and robust fluorescent pH nanosensor by co‐assembling oxidized DA (DA_ox) with a tripeptide GHK. The GHK‐DA_ox nanostructures have a quantum yield of 20.82%, which might be the brightest one among all the current co‐assembling structures merely through unmodified biomolecules. We envision this approach could open a new avenue for not only hybrid nanostructure construction, but also may inspire the bioengineering of in vivo luminescent probes.     

NOS1-mediated macrophage and endothelial cell interaction in the progression of atherosclerosis

Atherosclerosis is a chronic inflammatory disease arising due to an imbalance in lipid metabolism and maladaptive immune response driven by the accumulation of cholesterol-laden macrophages in the artery wall. Interactions between monocytes/macrophages and endothelial cells play an essential role in the pathogenesis of atherosclerosis. In our current study, nitric oxide synthase 1 (NOS1)-derived nitric oxide (NO) has been identified as a regulator of macrophage and endothelial cell interaction. Oxidized LDL (OxLDL) activates NOS1, which results in the expression of CD40 ligand in macrophages. OxLDL-stimulated macrophages produce some soluble factors which increase the CD40 receptor expression in endothelial cells. This increases the interaction between the macrophages and endothelial cells, which leads to an increase in the inflammatory response. Inhibition of NOS1-derived NO might serve as an effective strategy to reduce foam cell formation and limit the extent of atherosclerotic plaque expansion.     

Linking of cytochrome P450cam and putidaredoxin by a co-ordination bridge

Cytochrome P450_cam (CYP101) catalyzes the oxidation of D(+)-camphor at the 5 position. The enzyme couples the reduction of dioxygen to the oxidation of the substrate. To transfer electrons from the reductant (NADH) to the cytochrome, two additional proteins are required. These are putidaredoxin (PdX) and putidaredoxin reductase (PdR). We have chemically linked a form of PdX with a histidine tag at the C-terminus to the P450. To accomplish this, we have modified cysteine 334 on P450 with a bipyridinyl group, and co-ordinated the C-terminal histidine tag of PdX by the addition of Ni²⁺ or Ru³⁺. The Ru³⁺ complex was the most stable. The non-linked system gave mostly 5-ketocamphor, a product of two consecutive hydroxylations, and H₂O₂, a product of 2-electron uncoupling. The Ni²⁺ complex gave both 5-exo-hydroxycamphor and 5-ketocamphor, but it also uncoupled. The Ru³⁺ complex gave a single product (5-exo-hydroxycamphor) and did not uncouple at the optimal PdR concentration. Our results are consistent with other studies of this system, in that strong binding of PdX to P450 is crucial for good coupling and for release of 5-exo-hydroxycamphor.     

Lysoglycerophospholipids in chronic inflammatory disorders: The PLA2/LPC and ATX/LPA axes

Lysophosphatidylcholine (LPC) and lysophosphatidic acid (LPA), the most prominent lysoglycerophospholipids, are emerging as a novel class of inflammatory lipids, joining thromboxanes, leukotrienes and prostaglandins with which they share metabolic pathways and regulatory mechanisms. Enzymes that participate in LPC and LPA metabolism, such as the phospholipase A₂ superfamily (PLA₂) and autotaxin (ATX, ENPP2), play central roles in regulating LPC and LPA levels and consequently their actions. LPC/LPA biosynthetic pathways will be briefly presented and LPC/LPA signaling properties and their possible functions in the regulation of the immune system and chronic inflammation will be reviewed. Furthermore, implications of exacerbated LPC and/or LPA signaling in the context of chronic inflammatory diseases, namely rheumatoid arthritis, multiple sclerosis, pulmonary fibrosis and hepatitis, will be discussed. This article is part of a Special Issue entitled Advances in Lysophospholipid Research.     

Disulfide reduction abolishes tissue factor cofactor function

Background

Tissue factor (TF), an in vivo initiator of blood coagulation, is a transmembrane protein and has two disulfides in the extracellular domain. The integrity of one cysteine pair, Cys186–Cys209, has been hypothesized to be essential for an allosteric “decryption” phenomenon, presumably regulating TF procoagulant function, which has been the subject of a lengthy debate. The conclusions of published studies on this subject are based on indirect evidences obtained by the use of reagents with potentially oxidizing/reducing properties.

Methods

The status of disulfides in recombinant TF_1–263 and natural placental TF in their non-reduced native and reduced forms was determined by mass-spectrometry. Functional assays were performed to assess TF cofactor function.

Results

In native proteins, all four cysteines of the extracellular domain of TF are oxidized. Reduced TF retains factor VIIa binding capacity but completely loses the cofactor function.

Conclusion

The reduction of TF disulfides (with or without alkylation) eliminates TF regulation of factor VIIa catalytic function in both membrane dependent FX activation and membrane independent synthetic substrate hydrolysis.

General significance

Results of this study advance our knowledge on TF structure/function relationships.     

Involvement of thioredoxin y2 in the preservation of leaf methionine sulfoxide reductase capacity and growth under high light

Methionine (Met) in proteins can be oxidized to two diastereoisomers of methionine sulfoxide, Met‐S‐O and Met‐R‐O, which are reduced back to Met by two types of methionine sulfoxide reductases (MSRs), A and B, respectively. MSRs are generally supplied with reducing power by thioredoxins. Plants are characterized by a large number of thioredoxin isoforms, but those providing electrons to MSRs in vivo are not known. Three MSR isoforms, MSRA4, MSRB1 and MSRB2, are present in Arabidopsis thaliana chloroplasts. Under conditions of high light and long photoperiod, plants knockdown for each plastidial MSR type or for both display reduced growth. In contrast, overexpression of plastidial MSRBs is not associated with beneficial effects in terms of growth under high light. To identify the physiological reductants for plastidial MSRs, we analyzed a series of mutants deficient for thioredoxins f, m, x or y. We show that mutant lines lacking both thioredoxins y1 and y2 or only thioredoxin y2 specifically display a significantly reduced leaf MSR capacity (–25%) and growth characteristics under high light, related to those of plants lacking plastidial MSRs. We propose that thioredoxin y2 plays a physiological function in protein repair mechanisms as an electron donor to plastidial MSRs in photosynthetic organs.     

Antioxidative responses in Vitis vinifera infected by grapevine fanleaf virus

The antioxidative response of grapevine leaves (Vitis vinifera cv. Trebbiano) affected by the presence of grapevine fanleaf virus was studied during the summer of 2010 at three different harvest times (July 1st and 26th, and August 30th). At the first and second harvest, infected leaves showed increases in the concentration of superoxide radical and hydrogen peroxide, the latter increasing for enhanced activity of superoxide dismutase. In contrast, at the last harvest time, increases in the ascorbate pool and ascorbate peroxidase activity maintained hydrogen peroxide to control levels. The glutathione pool was negatively affected as summer progressed, showing a decrease in its total and reduced form amounts. At the same time, increases in the ascorbate pool were observed, making antioxidant defenses of grapevine effective also at the last harvest time. Increases in phenolic acids, and in particular in p-hydroxybenzoic acid, at the first and second harvest might have enhanced the efficiency of the antioxidant system through an interrelation between a peroxidase/phenol/ascorbate system and the NADPH/glutathione/ascorbate cycle. The lack of increase in p-hydroxybenzoic acid at the third harvest could be due instead to the enhanced utilization of this acid for hydrogen peroxide detoxification. With time, grapevine plants lost their capacity to contrast the spread of grapevine fanleaf virus, but acquired a greater ability to counteract pathogen-induced oxidative stress, being endowed with more reduced antioxidant pools.