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     

Discovery of a small-molecule inhibitor and cellular probe of Keap1–Nrf2 protein–protein interaction

A high-throughput screen (HTS) of the MLPCN library using a homogenous fluorescence polarization assay identified a small molecule as a first-in-class direct inhibitor of Keap1–Nrf2 protein–protein interaction. The HTS hit has three chiral centers; a combination of flash and chiral chromatographic separation demonstrated that Keap1-binding activity resides predominantly in one stereoisomer (SRS)-5 designated as ML334 (LH601A), which is at least 100× more potent than the other stereoisomers. The stereochemistry of the four cis isomers was assigned using X-ray crystallography and confirmed using stereospecific synthesis. (SRS)-5 is functionally active in both an ARE gene reporter assay and an Nrf2 nuclear translocation assay. The stereospecific nature of binding between (SRS)-5 and Keap1 as well as the preliminary but tractable structure–activity relationships support its use as a lead for our ongoing optimization     

Evaluation of bifidobacterial adhesion to acidic sugar chains of porcine colonic mucins

The aim of this study was to assess the adhesion of Bifidobacterium strains to acidic carbohydrate moieties of porcine colonic mucin. Mucins were extracted and purified via gel filtration chromatography followed by density-gradient ultracentrifugation. The presence of sulfated and sialylated carbohydrates in mucins was shown by enzyme-linked immunosorbent assays using PGM34 and HMC31 monoclonal antibodies (mAbs), respectively. Adhesion of Bifidobacterium strains to mucin preparations was markedly affected by the degree of purification. In eight of 22 strains, we observed increased adhesion to mucin preparations purified by ultracentrifugation. Moreover, in some of these eight strains, adhesion to mucin was reduced by pretreatment with sulfatase and/or sialidase, and competitively inhibited by pretreatment with PGM34 and/or HCM31 mAbs. Our results showed that some Bifidobacterium strains adhered to sulfo- and/or sialomucin and were able to recognize carbohydrate structures of the mAbs epitopes.     

EcoBalance 2018—Nexus of ideas: innovation by linking through life cycle thinking (9–12 October 2018, Tokyo,Japan)

The International Journal of Life Cycle Assessment -     

Cloning and expression analysis of a MAPKKK gene and a novel nodulin gene of Lotus japonicus

We isolated a cDNA encoding mitogen-activated protein kinase kinase kinase alpha, designated LjM3Kalpha, from Lotus japonicus, a model legume. The gene was expressed constitutively in roots, root nodules, and shoots. We also identified a novel nodulin gene, LjNUF, that shows specific expression in nodules. LjNUF resembles the C-terminal half of a hypothetical protein (pir//D85436), the N-terminal half of which is similar to a portion of mitogen-activated protein kinase kinase kinase gamma. Although LjNUF was predicted to be a secreted protein, its function remains to be clarified.     

Dual functions of fractalkine/CX3C ligand 1 in trafficking of perforin+/granzyme B+ cytotoxic effector lymphocytes that are defined by CX3CR1 expression

Fractalkine/CX3C ligand 1 and its receptor CX3CR1 are known to mediate both cell adhesion and cell migration. Here we show that CX3CR1 defines peripheral blood cytotoxic effector lymphocytes commonly armed with intracellular perforin and granzyme B, which include NK cells, gammadelta T cells, and terminally differentiated CD8(+) T cells. In addition, soluble fractalkine preferentially induced migration of cytotoxic effector lymphocytes. Furthermore, interaction of cytotoxic effector lymphocytes with membrane-bound fractalkine promoted subsequent migration to the secondary chemokines, such as macrophage inflammatory protein-1beta/CC ligand 4 or IL-8/CXC ligand 8. Thus, fractalkine expressed on inflamed endothelium may function as a vascular regulator for cytotoxic effector lymphocytes, regardless of their lineage and mode of target cell recognition, through its ability to capture them from blood flow and to promote their emigration in response to other chemokines.     

A novel mutation of desert hedgehog in a patient with 46,XY partial gonadal dysgenesis accompanied by minifascicular neuropathy

We describe a patient with 46,XY partial gonadal dysgenesis (PGD) who presented with polyneuropathy. Sural nerve pathology revealed peculiar findings characterized by extensive minifascicular formation within the endoneurium and with a decreased density of myelinated fibers. We found, in the patient, a homozygous missense mutation (ATG-->ACG) at the initiating codon in exon 1 of the desert hedgehog (DHH) gene, which predicts a failure of translation of the gene. The same heterozygous mutation was found in the patient's father. This is the first report of a human DHH gene mutation, and the findings demonstrate that mutation of the DHH gene may cause 46, XY PGD associated with minifascicular neuropathy.