Successful selective production of vanillic acid from depolymerized sulfite lignin and its application to poly(ethylene vanillate) synthesis.

Towards lignin upgrading, vanillic acid (VA), a lignin-derived guaiacyl compound, was produced from sulfite lignin for successfully synthesizing poly(ethylene vanillate), an aromatic polymer. The engineered Sphingobium sp. SYK-6-based strain in which the genes responsible for VA/3-O-methyl gallic acid O-demethylase and syringic acid O-demethylase were disrupted was able to produce vanillic acid (VA) from the mixture consisting of acetovanillone, vanillin, VA, and other low-molecular-weight aromatics obtained by Cu(OH)2-catalyzed alkaline depolymerization of sulfite lignin and membrane fractionation. From the bio-based VA, methyl-4-(2-hydroxyethoxy)-3-methoxybenzoate was synthesized via methylesterification, hydroxyethylation, and distillation, and then it was subjected to polymerization catalyzed by titanium tetraisopropoxide. The molecular weight of the obtained poly(ethylene vanillate) was evaluated to be Mw = 13,000 (Mw/Mn = 1.99) and its melting point was 261 °C. The present work proved that poly(ethylene vanillate) is able to be synthesized using VA produced from lignin for the first time.

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.

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.

Regulation of vanillate and syringate catabolism by a MarR-type transcriptional regulator DesR in Sphingobium sp. SYK-6.

Vanillate and syringate are major intermediate metabolites generated during the microbial degradation of lignin. In Sphingobium sp. SYK-6, vanillate is O demethylated to protocatechuate by LigM; protocatechuate is then catabolized via the protocatechuate 4,5-cleavage pathway. Syringate is O demethylated to gallate by consecutive reactions catalyzed by DesA and LigM, and then gallate is subjected to ring cleavage by DesB. Here, we investigated the transcriptional regulation of desA, ligM, and desB involved in vanillate and syringate catabolism. Quantitative reverse transcription-PCR analyses indicated that the transcription of these genes was induced 5.8-37-fold in the presence of vanillate and syringate. A MarR-type transcriptional regulator, SLG_12870 (desR), was identified as the gene whose product bound to the desB promoter region. Analysis of a desR mutant indicated that the transcription of desB, ligM, and desR is negatively regulated by DesR. Purified DesR bound to the upstream regions of desB, ligM, and desR, and the inverted repeat sequences similar to each other in these regions were suggested to be essential for DNA binding of DesR. Vanillate and syringate inhibited DNA binding of DesR, indicating that these compounds are effector molecules of DesR. The transcription of desA was found to be regulated by an as-yet unidentified regulator.

Enabling microbial syringol conversion through structure-guided protein engineering.

Microbial conversion of aromatic compounds is an emerging and promising strategy for valorization of the plant biopolymer lignin. A critical and often rate-limiting reaction in aromatic catabolism is O-aryl-demethylation of the abundant aromatic methoxy groups in lignin to form diols, which enables subsequent oxidative aromatic ring-opening. Recently, a cytochrome P450 system, GcoAB, was discovered to demethylate guaiacol (2-methoxyphenol), which can be produced from coniferyl alcohol-derived lignin, to form catechol. However, native GcoAB has minimal ability to demethylate syringol (2,6-dimethoxyphenol), the analogous compound that can be produced from sinapyl alcohol-derived lignin. Despite the abundance of sinapyl alcohol-based lignin in plants, no pathway for syringol catabolism has been reported to date. Here we used structure-guided protein engineering to enable microbial syringol utilization with GcoAB. Specifically, a phenylalanine residue (GcoA-F169) interferes with the binding of syringol in the active site, and on mutation to smaller amino acids, efficient syringol O-demethylation is achieved. Crystallography indicates that syringol adopts a productive binding pose in the variant, which molecular dynamics simulations trace to the elimination of steric clash between the highly flexible side chain of GcoA-F169 and the additional methoxy group of syringol. Finally, we demonstrate in vivo syringol turnover in Pseudomonas putida KT2440 with the GcoA-F169A variant. Taken together, our findings highlight the significant potential and plasticity of cytochrome P450 aromatic O-demethylases in the biological conversion of lignin-derived aromatic compounds.

Sequence similarity searches for morphine biosynthesis enzymes in bacteria yield putative targets for understanding associations between infection and opiate administration.

Exploiting the immunosuppressive, analgesic and highly addictive properties of morphine could increase the success of a bacterial pathogen. Therefore, we performed sequence similarity searches for two morphine biosynthesis demethylases in bacteria. For thebaine 6-O-demethylase and codeine O-demethylase, we found strong alignments to three (Pseudomonas aeruginosa, Klebsiella pneumoniae and Acinetobacter baumannii) of the six ESKAPE pathogens (Enterococcus faecalis, Staphylococcus aureus, K. pneumoniae, A. baumannii, P. aeruginosa and Enterobacter species) that are commonly associated with drug resistance and nosocomial infections. Expression of the aligned sequence found in P. aeruginosa (NP_252880.1/PA4191) is upregulated in isolates obtained from cystic fibrosis patients. Our findings provide putative mechanistic targets for understanding the role of morphine in pathogenicity.

Multiprotein Dynamic Combinatorial Chemistry: A Strategy for the Simultaneous Discovery of Subfamily-Selective Inhibitors for Nucleic Acid Demethylases FTO and ALKBH3.

Dynamic combinatorial chemistry (DCC) is a powerful supramolecular approach for discovering ligands for biomolecules. To date, most, if not all, biologically templated DCC systems employ only a single biomolecule to direct the self-assembly process. To expand the scope of DCC, herein, a novel multiprotein DCC strategy has been developed that combines the discriminatory power of a zwitterionic "thermal tag" with the sensitivity of differential scanning fluorimetry. This strategy is highly sensitive and could differentiate the binding of ligands to structurally similar subfamily members. Through this strategy, it was possible to simultaneously identify subfamily-selective probes against two clinically important epigenetic enzymes: FTO (7; IC50 =2.6 µm) and ALKBH3 (8; IC50 =3.7 µm). To date, this is the first report of a subfamily-selective ALKBH3 inhibitor. The developed strategy could, in principle, be adapted to a broad range of proteins; thus it is of broad scientific interest.

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.

Overcoming a hemihedral twinning problem in tetrahydrofolate-dependent O-demethylase crystals by the microseeding method.

A tetrahydrofolate-dependent O-demethylase, LigM, from Sphingobium sp. SYK-6 was crystallized by the hanging-drop vapour-diffusion method. However, the obtained P3121 or P3221 crystals, which diffracted to 2.5-3.3 Šresolution, were hemihedrally twinned. To overcome the twinning problem, microseeding using P3121/P3221 crystals as microseeds was performed with optimization of the reservoir conditions. As a result, another crystal form was obtained. The newly obtained crystal diffracted to 2.5-3.0 Šresolution and belonged to space group P21212, with unit-cell parameters a = 102.0, b = 117.3, c = 128.1 Å. The P21212 crystals diffracted to better than 2.0 Šresolution after optimizing the cryoconditions. Phasing using the single anomalous diffraction method was successful at 3.0 Šresolution with a Pt-derivative crystal. This experience suggested that microseeding is an effective method to overcome the twinning problem, even when twinned crystals are utilized as microseeds.

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.

Papaverine 7-O-demethylase, a novel 2-oxoglutarate/Fe(2+)-dependent dioxygenase from opium poppy.

Opium poppy (Papaver somniferum) produces several pharmacologically important benzylisoquinoline alkaloids including the vasodilator papaverine. Pacodine and palaudine are tri-O-methylated analogs of papaverine, which contains four O-linked methyl groups. However, the biosynthetic origin of pacodine and palaudine has not been established. Three members of the 2-oxoglutarate/Fe(2+)-dependent dioxygenases (2ODDs) family in opium poppy display widespread O-dealkylation activity on several benzylisoquinoline alkaloids with diverse structural scaffolds, and two are responsible for the antepenultimate and ultimate steps in morphine biosynthesis. We report a novel 2ODD from opium poppy catalyzing the efficient substrate- and regio-specific 7-O-demethylation of papaverine yielding pacodine. The occurrence of papaverine 7-O-demethylase (P7ODM) expands the enzymatic scope of the 2ODD family in opium poppy and suggests an unexpected biosynthetic route to pacodine.

[Bioinformatics analysis of DNA demethylase genes in Lonicera japonica Thunb].

The DNA demethylase genes are widespread in plants. Four DNA demethylase genes (LJDME1, LJDME2, LJDME3 and LJDME4) were obtained from transcriptome dataset of Lonicera japonica Thunb by using bioinformatics methods and the proteins' physicochemical properties they encoded were predicted. The phylogenetic tree showed that the four DNA demethylase genes and Arabidopsis thaliana DME had a close relationship. The result of gene expression model showed that four DNA demethylase genes were different between species. The expression levels of LJDME1 and LJDME2 were even more higher in Lonicera japonica var. chinensis than those in L. japonica. LJDME] and LJDME2 maybe regulate the active compounds of L. japonica. This study aims to lay a foundation for further understanding of the function of DNA demethylase genes in L. japonica.

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.

Conversion of phenyl methyl ethers by Desulfitobacterium spp. and screening for the genes involved.

Microbial growth coupled to O-demethylation of phenyl methyl ethers, which are lignin decomposition products, was described for acetogenic bacteria and recently also for two species belonging to the nonacetogenic genus Desulfitobacterium. To elucidate the potential role of desulfitobacteria in the O-demethylation of phenyl methyl ethers in the environment, we cultivated Desulfitobacterium chlororespirans, D. dehalogenans, D. metallireducens, and different strains of D. hafniense with phenyl methyl ethers as sole electron donors. With the exception of D. metallireducens, all species and strains tested were able to demethylate at least three of the four phenyl methyl ethers applied with fumarate, nitrate, or thiosulfate as electron acceptor. Furthermore, a high number of operons putatively encoding demethylase systems were identified in the genomes of Desulfitobacterium spp., although discrimination between O-, S-, N- and, Cl-demethylases was not possible. These findings provide evidence for the importance of the methylotrophic metabolism for desulfitobacteria and point to their involvement in the O-demethylation of phenyl methyl ethers in the environment.

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.

Corrinoid activation by a RACE protein: studies on the interaction of the proteins involved.

The O-demethylases of anaerobes are corrinoid-dependent, ether-cleaving methyltransferase enzyme systems consisting of four components. The interaction of the O-demethylase components of the acetogenic bacterium Acetobacterium dehalogenans was studied by protein mobility on native PAGE, far-Western blot analysis and yeast two-hybrid screen. Using native PAGE and far-Western blot, the interaction of the activating enzyme (AE) with its substrate, the corrinoid protein (CP), could be observed. The interaction occurred with four different CPs of A. dehalogenans and a CP from Desulfitobacterium hafniense DCB-2, all involved in ether cleavage. In the corrinoid reduction assay, the AE reduced all CPs tested. This result indicates a broad substrate specificity of the AE of A. dehalogenans. In addition, an interaction of the A. dehalogenans CP of the vanillate-O-demethylase with the two methyltransferases of the same enzyme system was observed. The interaction of the ether-cleaving methyltransferase with the CP appeared to be significantly less pronounced than that reported for the homologous methanol and methylamine methyltransferase systems of methanogenic archaea.

Origin of pisatin demethylase (PDA) in the genus Fusarium.

Host specificity of plant pathogens can be dictated by genes that enable pathogens to circumvent host defenses. Upon recognition of a pathogen, plants initiate defense responses that can include the production of antimicrobial compounds such as phytoalexins. The pea pathogen Nectria haematococca mating population VI (MPVI) is a filamentous ascomycete that contains a cluster of genes known as the pea pathogenicity (PEP) cluster in which the pisatin demethylase (PDA) gene resides. The PDA gene product is responsible for the detoxification of the phytoalexin pisatin, which is produced by the pea plant (Pisum sativum L.). This detoxification activity allows the pathogen to evade the phytoalexin defense mechanism. It has been proposed that the evolution of PDA and the PEP cluster reflects horizontal gene transfer (HGT). Previous observations consistent with this hypothesis include the location of the PEP cluster and PDA gene on a dispensable portion of the genome (a supernumerary chromosome), a phylogenetically discontinuous distribution of the cluster among closely related species, and a bias in G+C content and codon usage compared to other regions of the genome. In this study we compared the phylogenetic history of PDA, beta-tubulin, and translation elongation factor 1-alpha in three closely related fungi (Nectria haematococca, Fusarium oxysporum, and Neocosmospora species) to formally evaluate hypotheses regarding the origin and evolution of PDA. Our results, coupled with previous work, robustly demonstrate discordance between the gene genealogy of PDA and the organismal phylogeny of these species, and illustrate how HGT of pathogenicity genes can contribute to the expansion of host specificity in plant-pathogenic fungi.

Characterization of an O-demethylase of Desulfitobacterium hafniense DCB-2.

Besides acetogenic bacteria, only Desulfitobacterium has been described to utilize and cleave phenyl methyl ethers under anoxic conditions; however, no ether-cleaving O-demethylases from the latter organisms have been identified and investigated so far. In this study, genes of an operon encoding O-demethylase components of Desulfitobacterium hafniense strain DCB-2 were cloned and heterologously expressed in Escherichia coli. Methyltransferases I and II were characterized. Methyltransferase I mediated the ether cleavage and the transfer of the methyl group to the superreduced corrinoid of a corrinoid protein. Desulfitobacterium methyltransferase I had 66% identity (80% similarity) to that of the vanillate-demethylating methyltransferase I (OdmB) of Acetobacterium dehalogenans. The substrate spectrum was also similar to that of the latter enzyme; however, Desulfitobacterium methyltransferase I showed a higher level of activity for guaiacol and used methyl chloride as a substrate. Methyltransferase II catalyzed the transfer of the methyl group from the methylated corrinoid protein to tetrahydrofolate. It also showed a high identity (∼70%) to methyltransferases II of A. dehalogenans. The corrinoid protein was produced in E. coli as cofactor-free apoprotein that could be reconstituted with hydroxocobalamin or methylcobalamin to function in the methyltransferase I and II assays. Six COG3894 proteins, which were assumed to function as activating enzymes mediating the reduction of the corrinoid protein after an inadvertent oxidation of the corrinoid cofactor, were studied with respect to their abilities to reduce the recombinant reconstituted corrinoid protein. Of these six proteins, only one was found to catalyze the reduction of the corrinoid protein.