A methyl esterase 1 (PvMES1) promotes the salicylic acid pathway and enhances Fusarium wilt resistance in common beans.

KEY MESSAGE: Methyl esterase (MES), PvMES1, contributes to the defense response toward Fusarium wilt in common beans by regulating the salicylic acid (SA) mediated signaling pathway from phenylpropanoid synthesis and sugar metabolism as well as others. Common bean (Phaseolus vulgaris L.) is an important food legume. Fusarium wilt caused by Fusarium oxysporum f. sp. phaseoli is one of the most serious soil-borne diseases of common bean found throughout the world and affects the yield and quality of the crop. Few sources of Fusarium wilt resistance exist in legumes and most are of quantitative inheritance. In this study, we have identified a methyl esterase (MES), PvMES1, that contributes to plant defense response by regulating the salicylic acid (SA) mediated signaling pathway in response to Fusarium wilt in common beans. The result showed the role of PvMES1 in regulating SA levels in common bean and thus the SA signaling pathway and defense response mechanism in the plant. Overexpression of the PvMES1 gene enhanced Fusarium wilt resistance; while silencing of the gene caused susceptibility to the diseases. RNA-seq analysis with these transiently modified plants showed that genes related to SA level changes included the following gene ontologies: (a) phenylpropanoid synthesis; (b) sugar metabolism; and (c) interaction between host and pathogen as well as others. These key signal elements activated the defense response pathway in common bean to Fusarium wilt. Collectively, our findings indicate that PvMES1 plays a pivotal role in regulating SA biosynthesis and signaling, and increasing Fusarium wilt resistance in common bean, thus providing novel insight into the practical applications of both SA and MES genes and pathways they contribute to for developing elite crop varieties with enhanced broad-spectrum resistance to this critical disease.

Thebaine is Selectively Demethylated by Thebaine 6-O-Demethylase and Codeine-3-O-demethylase at Distinct Binding Sites: A Computational Study.

Two homologous 2-oxoglutarate-dependent (ODD) nonheme enzymes thebaine 6-O-demethylase (T6ODM) and codeine-3-O-demethylase (CODM), are involved in the morphine biosynthesis pathway from thebaine, catalyzing the O-demethylation reaction with precise regioselectivity at C6 and C3 positions of thebaine respectively. We investigated the origin of the regioselectivity of these enzymes by combined molecular dynamics (MD) and quantum mechanics/molecular mechanics (QM/MM) calculations and found that Thebaine binds at the two distinct sites of T6ODM and CODM, which determines the regioselectivity of the enzymes. A remarkable oxo rotation is observed in the decarboxylation process. Starting from the closed pentacoordinate configuration, the C-terminal lid adopts an open conformation in the octahedral Fe(IV) = O complex to facilitate the subsequent demethylation. Phe241 and Phe311 stabilize the substrate in the binding pocket, while Arg219 acts as a gatekeeper residue to stabilize the substrate. Our results unravel the regioselectivity in 2-OG dependent nonheme enzymes and may shed light for exploring the substrate scope of these enzymes and developing novel biotechnology for morphine biosynthesis.

The Syringate O-Demethylase Gene of Sphingobium sp. Strain SYK-6 Is Regulated by DesX, while Other Vanillate and Syringate Catabolism Genes Are Regulated by DesR.

Syringate and vanillate are the major metabolites of lignin biodegradation. In Sphingobium sp. strain SYK-6, syringate is O demethylated to gallate by consecutive reactions catalyzed by DesA and LigM, and vanillate is O demethylated to protocatechuate by a reaction catalyzed by LigM. The gallate ring is cleaved by DesB, and protocatechuate is catabolized via the protocatechuate 4,5-cleavage pathway. The transcriptions of desA, ligM, and desB are induced by syringate and vanillate, while those of ligM and desB are negatively regulated by the MarR-type transcriptional regulator DesR, which is not involved in desA regulation. Here, we clarified the regulatory system for desA transcription by analyzing the IclR-type transcriptional regulator desX, located downstream of desA Quantitative reverse transcription (RT)-PCR analyses of a desX mutant indicated that the transcription of desA was negatively regulated by DesX. In contrast, DesX was not involved in the regulation of ligM and desB The ferulate catabolism genes (ferBA), under the control of a MarR-type transcriptional regulator, FerC, are located upstream of desA RT-PCR analyses suggested that the ferB-ferA-SLG_25010-desA gene cluster consists of the ferBA operon and the SLG_25010-desA operon. Promoter assays revealed that a syringate- and vanillate-inducible promoter is located upstream of SLG_25010. Purified DesX bound to this promoter region, which overlaps an 18-bp inverted-repeat sequence that appears to be essential for the DNA binding of DesX. Syringate and vanillate inhibited the DNA binding of DesX, indicating that the compounds are effector molecules of DesX.IMPORTANCE Syringate is a major degradation product in the microbial and chemical degradation of syringyl lignin. Along with other low-molecular-weight aromatic compounds, syringate is produced by chemical lignin depolymerization. Converting this mixture into value-added chemicals using bacterial metabolism (i.e., biological funneling) is a promising option for lignin valorization. To construct an efficient microbial lignin conversion system, it is necessary to identify and characterize the genes involved in the uptake and catabolism of lignin-derived aromatic compounds and to elucidate their transcriptional regulation. In this study, we found that the transcription of desA, encoding syringate O-demethylase in SYK-6, is regulated by an IclR-type transcriptional regulator, DesX. The findings of this study, combined with our previous results on desR (encoding a MarR transcriptional regulator that controls the transcription of ligM and desB), provide an overall picture of the transcriptional-regulatory systems for syringate and vanillate catabolism in SYK-6.

Single-base methylome profiling of the giant kelp Saccharina japonica reveals significant differences in DNA methylation to microalgae and plants.

Brown algae have convergently evolved plant-like body plans and reproductive cycles, which in plants are controlled by differential DNA methylation. This contribution provides the first single-base methylome profiles of haploid gametophytes and diploid sporophytes of a multicellular alga. Although only c. 1.4% of cytosines in Saccharina japonica were methylated mainly at CHH sites and characterized by 5-methylcytosine (5mC), there were significant differences between life-cycle stages. DNA methyltransferase 2 (DNMT2), known to efficiently catalyze tRNA methylation, is assumed to methylate the genome of S. japonica in the structural context of tRNAs as the genome does not encode any other DNA methyltransferases. Circular and long noncoding RNA genes were the most strongly methylated regulatory elements in S. japonica. Differential expression of genes was negatively correlated with DNA methylation with the highest methylation levels measured in both haploid gametophytes. Hypomethylated and highly expressed genes in diploid sporophytes included genes involved in morphogenesis and halogen metabolism. The data herein provide evidence that cytosine methylation, although occurring at a low level, is significantly contributing to the formation of different life-cycle stages, tissue differentiation and metabolism in brown algae.

Structure of aryl O-demethylase offers molecular insight into a catalytic tyrosine-dependent mechanism.

Some strains of soil and marine bacteria have evolved intricate metabolic pathways for using environmentally derived aromatics as a carbon source. Many of these metabolic pathways go through intermediates such as vanillate, 3-O-methylgallate, and syringate. Demethylation of these compounds is essential for downstream aryl modification, ring opening, and subsequent assimilation of these compounds into the tricarboxylic acid (TCA) cycle, and, correspondingly, there are a variety of associated aryl demethylase systems that vary in complexity. Intriguingly, only a basic understanding of the least complex system, the tetrahydrofolate-dependent aryl demethylase LigM from Sphingomonas paucimobilis, a bacterial strain that metabolizes lignin-derived aromatics, was previously available. LigM-catalyzed demethylation enables further modification and ring opening of the single-ring aromatics vanillate and 3-O-methylgallate, which are common byproducts of biofuel production. Here, we characterize aryl O-demethylation by LigM and report its 1.81-Å crystal structure, revealing a unique demethylase fold and a canonical folate-binding domain. Structural homology and geometry optimization calculations enabled the identification of LigM's tetrahydrofolate-binding site and protein-folate interactions. Computationally guided mutagenesis and kinetic analyses allowed the identification of the enzyme's aryl-binding site location and determination of its unique, catalytic tyrosine-dependent reaction mechanism. This work defines LigM as a distinct demethylase, both structurally and functionally, and provides insight into demethylation and its reaction requirements. These results afford the mechanistic details required for efficient utilization of LigM as a tool for aryl O-demethylation and as a component of synthetic biology efforts to valorize previously underused aromatic compounds.

In vivo fluorescence-based characterization of cytochrome P450 activity during embryonic development of zebrafish (Danio rerio).

Zebrafish (Danio rerio) early life-stages are increasingly gaining attention as an alternative model in both human and environmental toxicology. Whereas there is amble knowledge about the transcription of various cytochrome P450 isoforms, the level of information about functional implications is still limited. This study investigated the development of CYP2-dependent 7-methoxycoumarin-O-demethylase (MCOD) activity throughout the early zebrafish development from 5 to 118 h post-fertilization (hpf) via confocal laser scanning microscopy. Results demonstrate that zebrafish embryos exhibit constitutive MCOD activity from as early as 5.5 hpf. Characteristic spatiotemporal patterns were documented with MCOD activities localized in several tissues and organs, namely the cardiovascular system, the brain, the digestive system, and the urinary tract. The study thereby contributes to a better understanding of the development and functional role of CYP enzymes in zebrafish early life-stages.

Kinetics of dextromethorphan-O-demethylase activity and distribution of CYP2D in four commonly-used subcellular fractions of rat brain.

The purpose of this study was to compare the enzymatic kinetics and distribution of cytochrome P450 2D (CYP2D) among different rat brain subcellular fractions. Rat brains were used to prepare total membrane, crude mitochondrial, purified mitochondrial, and microsomal fractions, in addition to total homogenate. Michaelis-Menten kinetics of the brain CYP2D activity was estimated based on the conversion of dextromethorphan (DXM) to dextrorphan using UPLC-MS/MS. Protein levels of CYP2D and subcellular markers were determined by Western blot. Microsomal CYP2D exhibited high affinity and low capacity, compared with the mitochondrial CYP2D that had a much lower (∼50-fold) affinity but a higher (∼six-fold) capacity. The apparent CYP2D affinity and capacity of the crude mitochondria were in between those of the microsomes and purified mitochondria. Additionally, the CYP2D activity in the whole homogenate was much higher than that in the total membranes at higher DXM concentrations. A CYP2D immune-reactive band in the brain mitochondria appeared at a lower MW but had a much higher intensity than that in the microsomes. Mitochondrial brain CYP2D has a much higher capacity than its microsomal counterpart. Additionally, brain homogenate is more representative of the overall CYP2D activity than the widely-used total membrane fraction.

Isolation of a novel platform bacterium for lignin valorization and its application in glucose-free cis,cis-muconate production.

Microbial production of cis,cis-muconate (ccMA) from phenolic compounds obtained by chemical depolymerization of lignin is a promising approach to valorize lignin. Because microbial production requires a large amount of carbon and energy source, it is desirable to establish a ccMA-producing strain that utilizes lignin-derived phenols instead of general sources like glucose. We isolated Pseudomonas sp. strain NGC7 that grows well on various phenolic compounds derived from p-hydroxyphenyl, guaiacyl, and syringyl units of lignin. An NGC7 mutant of protocatechuate (PCA) 3,4-dioxygenase and ccMA cycloisomerase genes (NGC703) lost the ability to grow on vanillate and p-hydroxybenzoate but grew normally on syringate. Introduction of a plasmid carrying genes encoding PCA decarboxylase, flavin prenyltransferase, vanillate O-demethylase, and catechol 1,2-dioxygenase into NGC703 enabled production of 3.2 g/L ccMA from vanillate with a yield of 75% while growing on syringate. This strain also produced ccMA from birch lignin-derived phenols. All these results indicate the utility of NGC7 in glucose-free ccMA production.

Biochemical Characterization of AP Lyase and m6A Demethylase Activities of Human AlkB Homologue 1 (ALKBH1).

Alkbh1 is one of nine mammalian homologues of Escherichia coli AlkB, a 2-oxoglutarate-dependent dioxygenase that catalyzes direct DNA repair by removing alkyl lesions from DNA. Six distinct enzymatic activities have been reported for Alkbh1, including hydroxylation of variously methylated DNA, mRNA, tRNA, or histone substrates along with the cleavage of DNA at apurinic/apyrimidinic (AP) sites followed by covalent attachment to the 5'-product. The studies described here extend the biochemical characterization of two of these enzymatic activities using human ALKBH1: the AP lyase and 6-methyl adenine DNA demethylase activities. The steady-state and single-turnover kinetic parameters for ALKBH1 cleavage of AP sites in DNA were determined and shown to be comparable to those of other AP lyases. The α,ß-unsaturated aldehyde of the 5'-product arising from DNA cleavage reacts predominantly with C129 of ALKBH1, but secondary sites also generate covalent adducts. The 6-methyl adenine demethylase activity was examined with a newly developed assay using a methylation-sensitive restriction endonuclease, and the enzymatic rate was found to be very low. Indeed, the demethylase activity was less than half that of the AP lyase activity when ALKBH1 samples were assayed using identical buffer conditions. The two enzymatic activities were examined using a series of site-directed variant proteins, revealing the presence of distinct but partially overlapping active sites for the two reactions. We postulate that the very low 6-methyl adenine oxygenase activity associated with ALKBH1 is unlikely to represent the major function of the enzyme in the cell, while the cellular role of the lyase activity (including its subsequent covalent attachment to DNA) remains uncertain.

Carbon Source-Dependent Inducible Metabolism of Veratryl Alcohol and Ferulic Acid in Pseudomonas putida CSV86.

Pseudomonas putida CSV86 degrades lignin-derived metabolic intermediates, viz, veratryl alcohol, ferulic acid, vanillin, and vanillic acid, as the sole sources of carbon and energy. Strain CSV86 also degraded lignin sulfonate. Cell respiration, enzyme activity, biotransformation, and high-pressure liquid chromatography (HPLC) analyses suggest that veratryl alcohol and ferulic acid are metabolized to vanillic acid by two distinct carbon source-dependent inducible pathways. Vanillic acid was further metabolized to protocatechuic acid and entered the central carbon pathway via the ß-ketoadipate route after ortho ring cleavage. Genes encoding putative enzymes involved in the degradation were found to be present at fer, ver, and van loci. The transcriptional analysis suggests a carbon source-dependent cotranscription of these loci, substantiating the metabolic studies. Biochemical and quantitative real-time (qRT)-PCR studies revealed the presence of two distinct O-demethylases, viz, VerAB and VanAB, involved in the oxidative demethylation of veratric acid and vanillic acid, respectively. This report describes the various steps involved in metabolizing lignin-derived aromatic compounds at the biochemical level and identifies the genes involved in degrading veratric acid and the arrangement of phenylpropanoid metabolic genes as three distinct inducible transcription units/operons. This study provides insight into the bacterial degradation of lignin-derived aromatics and the potential of P. putida CSV86 as a suitable candidate for producing valuable products.IMPORTANCEPseudomonas putida CSV86 metabolizes lignin and its metabolic intermediates as a carbon source. Strain CSV86 displays a unique property of preferential utilization of aromatics, including for phenylpropanoids over glucose. This report unravels veratryl alcohol metabolism and genes encoding veratric acid O-demethylase, hitherto unknown in pseudomonads, thereby providing new insight into the metabolic pathway and gene pool for lignin degradation in bacteria. The biochemical and genetic characterization of phenylpropanoid metabolism makes it a prospective system for its application in producing valuable products, such as vanillin and vanillic acid, from lignocellulose. This study supports the immense potential of P. putida CSV86 as a suitable candidate for bioremediation and biorefinery.

Overexpression of DEMETER, a DNA demethylase, promotes early apical bud maturation in poplar.

The transition from active growth to dormancy is critical for the survival of perennial plants. We identified a DEMETER-like (CsDML) cDNA from a winter-enriched cDNA subtractive library in chestnut (Castanea sativa Mill.), an economically and ecologically important species. Next, we characterized this DNA demethylase and its putative ortholog in the more experimentally tractable hybrid poplar (Populus tremula × alba), under the signals that trigger bud dormancy in trees. We performed phylogenetic and protein sequence analysis, gene expression profiling, and 5-methyl-cytosine methylation immunodetection studies to evaluate the role of CsDML and its homolog in poplar, PtaDML6. Transgenic hybrid poplars overexpressing CsDML were produced and analysed. Short days and cold temperatures induced CsDML and PtaDML6. Overexpression of CsDML accelerated short-day-induced bud formation, specifically from Stages 1 to 0. Buds acquired a red-brown coloration earlier than wild-type plants, alongside with the up-regulation of flavonoid biosynthesis enzymes and accumulation of flavonoids in the shoot apical meristem and bud scales. Our data show that the CsDML gene induces bud formation needed for the survival of the apical meristem under the harsh conditions of winter.

Cloning, expression, and characterization of a four-component O-demethylase from human intestinal bacterium Eubacterium limosum ZL-II.

Eubacterium limosum ZL-II was described to convert secoisolariciresinol (SECO) to its demethylating product 4,4'-dihydroxyenterodiol (DHEND) under anoxic conditions. However, the reaction cascade remains unclear. Here, the O-demethylase being responsible for the conversion was identified and characterized. Nine genes encoding two methyltransferase-Is (MT-I), two corrinoid proteins (CP), two methyltransferase-IIs (MT-II), and three activating enzymes (AE) were screened, cloned, and expressed in Escherichia coli. Four of the nine predicted enzymes, including ELI_2003 (MT-I), ELI_2004 (CP), ELI_2005 (MT-II), and ELI_0370 (AE), were confirmed to constitute the O-demethylase in E. limosum ZL-II. The complete O-demethylase (combining the four components) reaction system was reconstructed in vitro. As expected, the demethylating products 3-demethyl-SECO and DHEND were both produced. During the reaction process, ELI_2003 (MT-I) initially catalyzed the transfer of methyl group from SECO to the corrinoid of ELI_2004 ([CoI]-CP), yielding demethylating products and [CH3-CoIII]-CP; then ELI_2005 (MT-II) mediated the transfer of methyl group from [CH3-CoIII]-CP to tetrahydrofolate, forming methyltetrahydrofolate and [CoI]-CP. Due to the low redox potential of [CoII]/[CoI], [CoI]-CP was oxidized to [CoII]-CP immediately in vitro, and ELI_0370 (AE) was responsible for catalyzing the reduction of [CoII]-CP to its active form [CoI]-CP. The active-site residues in ELI_2003, ELI_2005, and ELI_0370 were subsequently determined using molecular modeling combined with site-directed mutagenesis. To our knowledge, this is the first study on the identification and characterization of a four-component O-demethylase from E. limosum ZL-II, which will facilitate the development of method to artificial synthesis of related bioactive chemicals.

Identification of a unique 2-oxoglutarate-dependent flavone 7-O-demethylase completes the elucidation of the lipophilic flavone network in basil.

Small molecule demethylation is considered unusual in plants. Of the studied instances, the N-demethylation of nicotine is catalyzed by a Cyt P450 monooxygenase, while the O-dealkylation of alkaloids in Papaver somniferum is mediated by 2-oxoglutarate-dependent dioxygenases (2-ODDs). This report describes a 2-ODD regiospecifically catalyzing the 7-O-demethylation of methoxylated flavones in peltate trichomes of sweet basil (Ocimum basilicum L.). Three candidate 2-ODDs were identified in the basil trichome transcriptome database. Only the candidate designated ObF7ODM1 was found to be active with and highly specific for the proposed natural substrates, gardenin B and 8-hydroxysalvigenin. Of the characterized 2-ODDs, ObF7ODM1 is most closely related to O-demethylases from Papaver. The demethylase activity in trichomes from four basil chemotypes matches well with the abundance of ObF7ODM1 peptides and transcripts in the same trichome preparations. Treatment of basil plants with a 2-ODD inhibitor prohexadione-calcium significantly reduced the accumulation of 7-O-demethylated flavone nevadensin, confirming the involvement of a 2-ODD in its formation. Notably, the full-length open reading frame of ObF7ODM1 contains a second in-frame AUG codon 57 nucleotides downstream of the first translation initiation codon. Both AUG codons are recognized by bacterial translation machinery during heterologous gene expression. The N-truncated ObF7ODM1 is nearly inactive. The N-terminus essential for activity is unique to ObF7ODM1 and does not align with the sequences of other 2-ODDs. Further studies will reveal whether alternative translation initiation plays a role in regulating the O-demethylase activity in planta. Molecular identification of the flavone 7-O-demethylase completes the biochemical elucidation of the lipophilic flavone network in basil.

Diffusion of macromolecules in self-assembled cellulose/hemicellulose hydrogels.

Cellulose hydrogels are extensively applied in many biotechnological fields and are also used as models for plant cell walls. We synthesised model cellulosic hydrogels containing hemicelluloses, as a biomimetic of plant cell walls, in order to study the role of hemicelluloses on their mass transport properties. Microbial cellulose is able to self-assemble into composites when hemicelluloses, such as xyloglucan and arabinoxylan, are present in the incubation media, leading to hydrogels with different nano and microstructures. We investigated the diffusivities of a series of fluorescently labelled dextrans, of different molecular weight, and proteins, including a plant pectin methyl esterase (PME), using fluorescence recovery after photobleaching (FRAP). The presence of xyloglucan, known to be able to crosslink cellulose fibres, confirmed by scanning electron microscopy (SEM) and (13)C NMR, reduced mobility of macromolecules of molecular weight higher than 10 kDa, reflected in lower diffusion coefficients. Furthermore PME diffusion was reduced in composites containing xyloglucan, despite the lack of a particular binding motif in PME for this polysaccharide, suggesting possible non-specific interactions between PME and this hemicellulose. In contrast, hydrogels containing arabinoxylan coating cellulose fibres showed enhanced diffusivity of the molecules studied. The different diffusivities were related to the architectural features found in the composites as a function of polysaccharide composition. Our results show the effect of model hemicelluloses in the mass transport properties of cellulose networks in highly hydrated environments relevant to understanding the role of hemicelluloses in the permeability of plant cell walls and aiding design of plant based materials with tailored properties.

A collection of cytochrome P450 monooxygenase genes involved in modification and detoxification of herbicide atrazine in rice (Oryza sativa) plants.

Plant cytochrome P450 monooxygenases constitute one of the largest families of protein genes involved in plant growth, development and acclimation to biotic and abiotic stresses. However, whether these genes respond to organic toxic compounds and their biological functions for detoxifying toxic compounds such as herbicides in rice are poorly understood. The present study identified 201 genes encoding cytochrome P450s from an atrazine-exposed rice transcriptome through high-throughput sequencing. Of these, 69 cytochrome P450 genes were validated by microarray and some of them were confirmed by real time PCR. Activities of NADPH-cytochrome P450 reductase (CPR) and p-nitroanisole O-demethylase (PNOD) related to toxicity were determined and significantly induced by atrazine exposure. To dissect the mechanism underlying atrazine modification and detoxification by P450, metabolites (or derivatives) of atrazine in plants were analyzed by ultra performance liquid chromatography mass spectrometry (UPLC/MS). Major metabolites comprised desmethylatrazine (DMA), desethylatrazine (DEA), desisopropylatrazine (DIA), hydroxyatrazine (HA), hydroxyethylatrazine (HEA) and hydroxyisopropylatrazine (HIA). All of them were chemically modified by P450s. Furthermore, two specific inhibitors of piperonyl butoxide (PBO) and malathion (MAL) were used to assess the correlation between the P450s activity and rice responses including accumulation of atrazine in tissues, shoot and root growth and detoxification.

Three-Component O-Demethylase System Essential for Catabolism of a Lignin-Derived Biphenyl Compound in Sphingobium sp. Strain SYK-6.

Sphingobium sp. strain SYK-6 is able to assimilate lignin-derived biaryls, including a biphenyl compound, 5,5'-dehydrodivanillate (DDVA). Previously, ligXa (SLG_07770), which is similar to the gene encoding oxygenase components of Rieske-type nonheme iron aromatic-ring-hydroxylating oxygenases, was identified to be essential for the conversion of DDVA; however, the genes encoding electron transfer components remained unknown. Disruption of putative electron transfer component genes scattered through the SYK-6 genome indicated that SLG_08500 and SLG_21200, which showed approximately 60% amino acid sequence identities with ferredoxin and ferredoxin reductase of dicamba O-demethylase, were essential for the normal growth of SYK-6 on DDVA. LigXa and the gene products of SLG_08500 (LigXc) and SLG_21200 (LigXd) were purified and were estimated to be a trimer, a monomer, and a monomer, respectively. LigXd contains FAD as the prosthetic group and showed much higher reductase activity toward 2,6-dichlorophenolindophenol with NADH than with NADPH. A mixture of purified LigXa, LigXc, and LigXd converted DDVA into 2,2',3-trihydroxy-3'-methoxy-5,5'-dicarboxybiphenyl in the presence of NADH, indicating that DDVA O-demethylase is a three-component monooxygenase. This enzyme requires Fe(II) for its activity and is highly specific for DDVA, with a Km value of 63.5 µM and kcat of 6.1 s(-1). Genome searches in six other sphingomonads revealed genes similar to ligXc and ligXd (>58% amino acid sequence identities) with a limited number of electron transfer component genes, yet a number of diverse oxygenase component genes were found. This fact implies that these few electron transfer components are able to interact with numerous oxygenase components and the conserved LigXc and LigXd orthologs are important in sphingomonads.

Expression and substrate range of Streptomyces vanillate demethylase.

Vanillate is converted to protocatechuate by an O-demethylase consisting of VanA and VanB in Streptomyces sp. NL15-2K. In this study, vanillate demethylase from this strain was functionally expressed in Escherichia coli, and its substrate range for vanillate analogs was determined by an in vivo assay using recombinant whole cells. Among aromatic methyl ethers, vanillate, syringate, m-anisate, and veratrate were good substrates, whereas ferulate, vanillin, and guaiacol were not recognized by Streptomyces vanillate demethylase. After vanillate, 4-hydroxy-3-methylbenzoate was a better substrate than m-anisate and veratrate, and the 3-methyl group was efficiently oxidized to a hydroxymethyl group. These observations suggest that the combination of a carboxyl group on the benzene ring and a hydroxyl group in the para-position relative to the carboxyl group may be preferable for substrate recognition by the enzyme. (1)H-NMR analysis showed that the demethylation product of veratrate was isovanillate rather than vanillate. Therefore, it was concluded that O-demethylation of veratrate by Streptomyces vanillate demethylase occurred only at the meta-position relative to the carboxyl group.

Structure of the cobalamin-binding protein of a putative O-demethylase from Desulfitobacterium hafniense DCB-2.

This study describes the identification and the structural and spectroscopic analysis of a cobalamin-binding protein (termed CobDH) implicated in O-demethylation by the organohalide-respiring bacterium Desulfitobacterium hafniense DCB-2. The 1.5 Šresolution crystal structure of CobDH is presented in the cobalamin-bound state and reveals that the protein is composed of an N-terminal helix-bundle domain and a C-terminal Rossmann-fold domain, with the cobalamin coordinated in the base-off/His-on conformation similar to other cobalamin-binding domains that catalyse methyl-transfer reactions. EPR spectroscopy of CobDH confirms cobalamin binding and reveals the presence of a cob(III)alamin superoxide, indicating binding of oxygen to the fully oxidized cofactor. These data provide the first structural insights into the methyltransferase reactions that occur during O-demethylation by D. hafniense.

Global relevance of Aire binding to hypomethylated lysine-4 of histone-3.

Aire promotes the ectopic expression of a repertoire of peripheral-tissue antigens (PTAs) in thymic medullary epithelial cells (MECs) to mediate deletional tolerance and thereby prevent autoimmunity. Binding of hypomethylated histone 3 (H3)-tails by Aire's plant homeodomain (PHD) finger is essential for Aire function in cultured cell models, prompting speculation that Aire-PHD:H3-tail interactions underlie targeting of Aire to weakly transcribed loci. To evaluate the role of Aire's PHD finger in MECs on a global scale in vivo, we complemented Aire-deficient mice with a mutant of Aire that inhibits its binding to hypomethylated H3K4 residues. Although the range of Aire-targeted genes was largely unaffected in these mice, the D299A mutation caused a global dampening of Aire's transcriptional impact, resulting in an autoimmune disease similar in profile to that of their Aire-deficient counterparts. To test whether a low H3K4 methylation state is sufficient for Aire targeting, we overexpressed an H3K4-specific demethylase in an Aire-dependent cultured cell system, and determined its capacity to extend Aire's transcriptional footprint. The range and magnitude of Aire-regulated genes was largely unaffected, the only genes additionally induced by Aire in this context being those already accessed for repression. In short, Aire's H3-binding module is necessary for Aire-mediated regulation of gene expression and central tolerance induction, but this influence is unlikely to reflect a targeting mechanism solely based on the recognition of hypomethylated H3K4 residues.

Two potent cytochrome P450 2D6 inhibitors found in Rhodiola rosea.

OBJECTIVES: Throughout the world, in particular in Russia, Northern Europe and China, Rhodiola species are used as herb supplements. Previously, we found that the extract of Rhodiola rosea, one of the most widely used Rhodiola species, had an inhibitory effect on the catalytic activity of cytochrome P450 2D6. Here, its inhibitory components were identified. METHODS: A human liver microsomal in vitro system was used with dextromethorphan as substrate. The production rate of destrorphan, a metabolite of dextromethorphan, was used to measure enzyme activity. The concentration of destrorphan in the samples was measured using LC-MS/MS. Inhibitory activity of eight main components from Rhodiola rosea was evaluated. RESULTS: Rhodiosin and rhodionin showed inhibitory activity with IC50 values of 0.761 microM and 0.420 microM, respectively. The other components showed no obvious inhibition (with a residual enzyme activity of more than 90%). Both rhodiosin and rhodionin were determined to be non-competitive inhibitors with Ki values of 0.769 microM and 0.535 microM. CONCLUSION: Two of the main Rhodiola rosea compounds, rhodiosin and rhodionin, can inhibit cytochrome P450 2D6 non-competitively with high specificity which could have implications for interactions with co-administered drugs.