- Cfr and RlmN contain a single [4Fe-4S] cluster, which directs two distinct reactivities for s -adenosylmethionine: Methyl transfer by SN2 displacement and radical generation
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The radical SAM (RS) proteins RlmN and Cfr catalyze methylation of carbons 2 and 8, respectively, of adenosine 2503 in 23S rRNA. Both reactions are similar in scope, entailing the synthesis of a methyl group partially derived from S-adenosylmethionine (SAM) onto electrophilic sp2-hybridized carbon atoms via the intermediacy of a protein S-methylcysteinyl (mCys) residue. Both proteins contain five conserved Cys residues, each required for turnover. Three cysteines lie in a canonical RS CxxxCxxC motif and coordinate a [4Fe-4S]-cluster cofactor; the remaining two are at opposite ends of the polypeptide. Here we show that each protein contains only the one "radical SAM" [4Fe-4S] cluster and the two remaining conserved cysteines do not coordinate additional iron-containing species. In addition, we show that, while wild-type RlmN bears the C355 mCys residue in its as-isolated state, RlmN that is either engineered to lack the [4Fe-4S] cluster by substitution of the coordinating cysteines or isolated from Escherichia coli cultured under iron-limiting conditions does not bear a C355 mCys residue. Reconstitution of the [4Fe-4S] cluster on wild-type apo RlmN followed by addition of SAM results in rapid production of S-adenosylhomocysteine (SAH) and the mCys residue, while treatment of apo RlmN with SAM affords no observable reaction. These results indicate that in Cfr and RlmN, SAM bound to the unique iron of the [4Fe-4S] cluster displays two reactivities. It serves to methylate C355 of RlmN (C338 of Cfr), or to generate the 5′-deoxyadenosyl 5′-radical, required for substrate-dependent methyl synthase activity.
- Grove, Tyler L.,Radle, Matthew I.,Krebs, Carsten,Booker, Squire J.
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- A bacterial source for mollusk pyrone polyketides
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In the oceans, secondary metabolites often protect otherwise poorly defended invertebrates, such as shell-less mollusks, from predation. The origins of these metabolites are largely unknown, but many of them are thought to be made by symbiotic bacteria. In contrast, mollusks with thick shells and toxic venoms are thought to lack these secondary metabolites because of reduced defensive needs. Here, we show that heavily defended cone snails also occasionally contain abundant secondary metabolites, γ-pyrones known as nocapyrones, which are synthesized by symbiotic bacteria. The bacteria, Nocardiopsis alba CR167, are related to widespread actinomycetes that we propose to be casual symbionts of invertebrates on land and in the sea. The natural roles of nocapyrones are unknown, but they are active in neurological assays, revealing that mollusks with external shells are an overlooked source of secondary metabolite diversity.
- Lin, Zhenjian,Torres, Joshua P.,Ammon, Mary Anne,Marett, Lenny,Teichert, Russell W.,Reilly, Christopher A.,Kwan, Jason C.,Hughen, Ronald W.,Flores, Malem,Tianero, Ma. Diarey,Peraud, Olivier,Cox, James E.,Light, Alan R.,Villaraza, Aaron Joseph L.,Haygood, Margo G.,Concepcion, Gisela P.,Olivera, Baldomero M.,Schmidt, Eric W.
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- S-adenosyl-L-methionine: Anol-O-methyltransferase activity in organ cultures of Pimpinella anisum
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The biosynthesis of epoxypseudoisoeugenol-2-methylbutyrate (EPB), a rare phenylpropanoid of the genus Pimpinella, was investigated in vitro by means of a leaf-differentiating callus culture of Pimpinella anisum. In an effort to corroborate an earlier proposed biosynthetic pathway of EPB, the step between p-coumaryl alcohol and (E)-anethole was reinvestigated. Further feeding experiments with 14C-labelled precursors and preliminary studies at the enzyme level clearly revealed that anol, and not p-methoxycinnamyl alcohol as previously asserted, is an obligatory intermediate in EPB biosynthesis. S-Adenosyl-L-methionine: anol-O-methyltransferase, a key enzyme in EPB biosynthesis, was demonstrated and characterized for the first time.
- Kemmerer, Birgitt,Reichling, Juergen
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- Mechanistic studies on transcriptional coactivator protein arginine methyltransferase 1
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Protein arginine methyltransferases (PRMTs) catalyze the transfer of methyl groups from S-adenosylmethionine (SAM) to the guanidinium group of arginine residues in a number of important cell signaling proteins. PRMT1 is the founding member of this family, and its activity appears to be dysregulated in heart disease and cancer. To begin to characterize the catalytic mechanism of this isozyme, we assessed the effects of mutating a number of highly conserved active site residues (i.e., Y39, R54, E100, E144, E153, M155, and H293), which are believed to play key roles in SAM recognition, substrate binding, and catalysis. The results of these studies, as well as pH-rate studies, and the determination of solvent isotope effects (SIEs) indicate that M155 plays a critical role in both SAM binding and the processivity of the reaction but is not responsible for the regiospecific formation of asymmetrically dimethylated arginine (ADMA). Additionally, mutagenesis studies on H293, combined with pH studies and the lack of a normal SIE, do not support a role for this residue as a general base. Furthermore, the lack of a normal SIE with either the wild type or catalytically impaired mutants suggests that general acid/base catalysis is not important for promoting methyl transfer. This result, combined with the fact that the E144A/E153A double mutant retains considerably more activity then the single mutants alone, suggests that the PRMT1-catalyzed reaction is primarily driven by bringing the substrate guanidinium into the proximity of the S-methyl group of SAM and that the prior deprotonation of the substrate guanidinium is not required for methyl transfer.
- Rust, Heather L.,Zurita-Lopez, Cecilia I.,Clarke, Steven,Thompson, Paul R.
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- Expression and purification of an ArsM-elastin-like polypeptide fusion and its enzymatic properties
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Enzymes could act as a useful tool for environmental bioremediation. Arsenic (As) biomethylation, which can convert highly toxic arsenite [As(III)] into low-toxic volatile trimethylarsine, is considered to be an effective strategy for As removal from contaminated environments. As(III) S-adenosylmethyltransferase (ArsM) is a key enzyme for As methylation; its properties and preparation are crucial for its wide application. Currently, ArsM is usually purified as a His-tag fusion protein restricting widespread use due to high costs. In this study, to greatly reduce the cost and simplify the ArsM preparation process, an Elastin-like polypeptide (ELP) tag was introduced to construct an engineered Escherichia coli (ArsM-ELP). Consequently, a cost-effective and simple non-chromatographic purification approach could be used for ArsM purification. The enzymatic properties of ArsM-ELP were systematically investigated. The results showed that the As methylation rate of purified ArsM-ELP (> 35.49%) was higher than that of E. coli (ArsM-ELP) (> 10.39%) when exposed to 25?μmol/L and 100?μmol/L As(III), respectively. The purified ArsM-ELP was obtained after three round inverse transition cycling treatment in 2.0?mol/L NaCl at 32?°C for 10?min with the yield reaching more than 9.6% of the total protein. The optimal reaction temperature, pH, and time of ArsM-ELP were 30?°C, 7.5 and 30?min, respectively. The enzyme activity was maintained at over 50% at 45?°C for 12?h. The enzyme specific activity was 438.8 ± 2.1?U/μmol. ArsM-ELP had high selectivity for As(III). 2-Mercaptoethanol could promote enzyme activity, whereas SDS, EDTA, Fe2+, and Cu2+ inhibited enzyme activity, and Mg2+, Zn2+, Ca2+, and K+ had no significant effects on it.
- Ke, Changdong,Xiong, Hui,Zhao, Chungui,Zhang, Zhigang,Zhao, Xiaolan,Rensing, Christopher,Zhang, Guangya,Yang, Suping
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- Characterizing DNA methyltransferases with an ultrasensitive luciferase-linked continuous assay
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DNA (cytosine-5)-methyltransferases (DNMTs) catalyze the transfer of a methyl group from S-adenosyl-l-methionine (AdoMet) to the 5-position of cytosine residues and thereby silence transcription of regulated genes. DNMTs are important epigenetic targets. However, isolated DNMTs are weak catalysts and are difficult to assay. We report an ultrasensitive luciferase-linked continuous assay that converts the S-adenosyl-l-homocysteine product of DNA methylation to a quantifiable luminescent signal. Results with this assay are compared with the commonly used DNA labeling from [methyl-3H]AdoMet. A 5′-methylthioadenosine-adenosylhomocysteine nucleosidase is used to hydrolyze AdoHcy to adenine. Adenine phosphoribosyl transferase converts adenine to AMP and pyruvate orthophosphate dikinase converts AMP to ATP. Firefly luciferase gives a stable luminescent signal that results from continuous AMP recycling to ATP. This assay exhibits a broad dynamic range (0.1-1000 pmol of AdoHcy). The rapid response time permits continuous assays of DNA methylation detected by light output. The assay is suitable for high-throughput screening of chemical libraries for DNMT inhibition activity. The kinetic properties of human and bacterial CpG methyltransferases are characterized using this assay. Human catalytic domain DNMT3b activation by DNMT3L is shown to involve two distinct kinetic states that alter kcat but not Km for AdoMet. The assay is shown to be robust in the presence of high concentrations of the pyrimidine analogues 5-azacytidine and 5-azacytosine.
- Hemeon, Ivan,Gutierrez, Jemy A.,Ho, Meng-Chiao,Schramm, Vern L.
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- Three-Dimensional Proteome-Wide Scale Screening for the 5-Alpha Reductase Inhibitor Finasteride: Identification of a Novel Off-Target
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Finasteride, a 5-alpha reductase (5α-R) inhibitor, is a widely used drug for treating androgen-dependent conditions. However, its use is associated with sexual, psychological, and physical complaints, suggesting that other mechanisms, in addition to 5α-R inhibition, may be involved. Here, a multidisciplinary approach has been used to identify potential finasteride off-target proteins. SPILLO-PBSS software suggests an additional inhibitory activity of finasteride on phenylethanolamine N-methyltransferase (PNMT), the limiting enzyme in formation of the stress hormone epinephrine. The interaction of finasteride with PNMT was supported by docking and molecular dynamics analysis and by in vitro assay, confirming the inhibitory nature of the binding. Finally, this inhibition was also confirmed in an in vivo rat model. Literature data indicate that PNMT activity perturbation may be correlated with sexual and psychological side effects. Therefore, results here obtained suggest that the binding of finasteride to PNMT might have a role in producing the side effects exerted by finasteride treatment.
- Giatti, Silvia,Di Domizio, Alessandro,Diviccaro, Silvia,Falvo, Eva,Caruso, Donatella,Contini, Alessandro,Melcangi, Roberto Cosimo
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p. 4553 - 4566
(2021/05/06)
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- Structural Insights into the Mechanism of the Radical SAM Carbide Synthase NifB, a Key Nitrogenase Cofactor Maturating Enzyme
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Nitrogenase is a key player in the global nitrogen cycle, as it catalyzes the reduction of dinitrogen into ammonia. The active site of the nitrogenase MoFe protein corresponds to a [MoFe7S9C-(R)-homocitrate] species designated FeMo-cofactor, whose biosynthesis and insertion requires the action of over a dozen maturation proteins provided by the NIF (for NItrogen Fixation) assembly machinery. Among them, the radical SAM protein NifB plays an essential role, concomitantly inserting a carbide ion and coupling two [Fe4S4] clusters to form a [Fe8S9C] precursor called NifB-co. Here we report on the X-ray structure of NifB from Methanotrix thermoacetophila at 1.95 ? resolution in a state pending the binding of one [Fe4S4] cluster substrate. The overall NifB architecture indicates that this enzyme has a single SAM binding site, which at this stage is occupied by cysteine residue 62. The structure reveals a unique ligand binding mode for the K1-cluster involving cysteine residues 29 and 128 in addition to histidine 42 and glutamate 65. The latter, together with cysteine 62, belongs to a loop inserted in the active site, likely protecting the already present [Fe4S4] clusters. These two residues regulate the sequence of events, controlling SAM dual reactivity and preventing unwanted radical-based chemistry before the K2 [Fe4S4] cluster substrate is loaded into the protein. The location of the K1-cluster, too far away from the SAM binding site, supports a mechanism in which the K2-cluster is the site of methylation.
- Echavarri-Erasun, Carlos,Fajardo, Ana Sosa,Legrand, Pierre,Martin, Lydie,Nicolet, Yvain,Payá-Tormo, Luc?a,Pellicer Mart?nez, Maria Teresa,Rubio, Luis M.,Vernède, Xavier
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supporting information
p. 11006 - 11012
(2020/07/13)
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- Biosynthesis of methyl (E)-cinnamate in the liverwort Conocephalum salebrosum and evolution of cinnamic acid methyltransferase
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Methyl (E)-cinnamate is a specialized metabolite that occurs in a variety of land plants. In flowering plants, it is synthesized by cinnamic acid methyltransferase (CAMT)that belongs to the SABATH family. While rarely reported in bryophytes, methyl (E)-cinnamate is produced by some liverworts of the Conocephalum conicum complex, including C. salebrosum. In axenically grown thalli of C. salebrosum, methyl (E)-cinnamate was detected as the dominant compound. To characterize its biosynthesis, six full-length SABATH genes, which were designated CsSABATH1-6, were cloned from C. salebrosum. These six genes showed different levels of expression in the thalli of C. salebrosum. Next, CsSABATH1-6 were expressed in Escherichia coli to produce recombinant proteins, which were tested for methyltransferase activity with cinnamic acid and a few related compounds as substrates. Among the six SABATH proteins, CsSABATH6 exhibited the highest level of activity with cinnamic acid. It was renamed CsCAMT. The apparent Km value of CsCAMT using (E)-cinnamic acid as substrate was determined to be 50.5 μM. In contrast, CsSABATH4 was demonstrated to function as salicylic acid methyltransferase and was renamed CsSAMT. Interestingly, the CsCAMT gene from a sabinene-dominant chemotype of C. salebrosum is identical to that of the methyl (E)-cinnamate-dominant chemotype. Structure models for CsCAMT, CsSAMT and one flowering plant CAMT (ObCCMT1)in complex with (E)-cinnamic acid and salicylic acid were built, which provided structural explanations to substrate specificity of these three enzymes. In phylogenetic analysis, CsCAMT and ObCCMT1 were in different clades, implying that methyl (E)-cinnamate biosynthesis in bryophytes and flowering plants originated through convergent evolution.
- Zhang, Chi,Chen, Xinlu,Crandall-Stotler, Barbara,Qian, Ping,K?llner, Tobias G.,Guo, Hong,Chen, Feng
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- Reductive Cleavage of Sulfoxide and Sulfone by Two Radical S-Adenosyl- l -methionine Enzymes
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Sulfoxides and sulfones are commonly found in nature as a result of thioether oxidation, whereas only a very few enzymes have been found to metabolize these compounds. Utilizing the strong reduction potential of the [4Fe-4S] cluster of radical S-adenosyl-l-methionine (SAM) enzymes, we herein report the first enzyme-catalyzed reductive cleavage of sulfoxide and sulfone. We show two radical SAM enzymes, tryptophan lyase NosL and the class C radical SAM methyltransferase NosN, are able to act on a sulfoxide SAHO and a sulfone SAHO2, both of which are structurally similar to SAM. NosL cleaves all of the three bonds (i.e., S-C(5′), S-C(γ), and S-O) connecting the sulfur center of SAHO, with a preference for S-C(5′) bond cleavage. Similar S-C cleavage activity was also found for SHAO2, but no S-O cleavage was observed. In contrast to NosL, NosN almost exclusively cleaves the S-C(5′) bonds of SAHO and SAHO2 with much higher efficiencies. Our study provides valuable insights into the [4Fe-4S] cluster-mediated reduction reactions and highlights the remarkable catalytic promiscuity of radical SAM enzymes.
- Mandalapu, Dhanaraju,Ji, Xinjian,Zhang, Qi
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- Mechanism of a Class C Radical S-Adenosyl- l -methionine Thiazole Methyl Transferase
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The past decade has seen the discovery of four different classes of radical S-adenosylmethionine (rSAM) methyltransferases that methylate unactivated carbon centers. Whereas the mechanism of class A is well understood, the molecular details of methylation by classes B-D are not. In this study, we present detailed mechanistic investigations of the class C rSAM methyltransferase TbtI involved in the biosynthesis of the potent thiopeptide antibiotic thiomuracin. TbtI C-methylates a Cys-derived thiazole during posttranslational maturation. Product analysis demonstrates that two SAM molecules are required for methylation and that one SAM (SAM1) is converted to 5′-deoxyadenosine and the second SAM (SAM2) is converted to S-adenosyl-l-homocysteine (SAH). Isotope labeling studies show that a hydrogen is transferred from the methyl group of SAM2 to the 5′-deoxyadenosine of SAM1 and the other two hydrogens of the methyl group of SAM2 appear in the methylated product. In addition, a hydrogen appears to be transferred from the β-position of the thiazole to the methyl group in the product. We also show that the methyl protons in the product can exchange with solvent. A mechanism consistent with these observations is presented that differs from other characterized radical SAM methyltransferases.
- Zhang, Zhengan,Mahanta, Nilkamal,Hudson, Graham A.,Mitchell, Douglas A.,Van Der Donk, Wilfred A.
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p. 18623 - 18631
(2017/12/26)
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- Stereochemical Course of the Reaction Catalyzed by RimO, a Radical SAM Methylthiotransferase
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RimO is a member of the growing radical S-adenosylmethionine (SAM) superfamily of enzymes, which use a reduced [4Fe-4S] cluster to effect reductive cleavage of the 5′ C-S bond of SAM to form a 5′-deoxyadenosyl 5′-radical (5′-dA? intermediate. RimO uses this potent oxidant to catalyze the attachment of a methylthio group (-SCH3) to C3 of aspartate 89 of protein S12, one of 21 proteins that compose the 30S subunit of the bacterial ribosome. However, the exact mechanism by which this transformation takes place has remained elusive. Herein, we describe the stereochemical course of the RimO reaction. Using peptide mimics of the S12 protein bearing deuterium at the 3 pro-R or 3 pro-S positions of the target aspartyl residue, we show that RimO from Bacteroides thetaiotaomicron (Bt) catalyzes abstraction of the pro-S hydrogen atom, as evidenced by the transfer of deuterium into 5′-deoxyadenosine (5′-dAH). The observed kinetic isotope effect on H atom versus D atom abstraction is ～1.9, suggesting that this step is at least partially rate determining. We also demonstrate that Bt RimO can utilize the flavodoxin/flavodoxin oxidoreductase/NADPH reducing system from Escherichia coli as a source of requisite electrons. Use of this in vivo reducing system decreases, but does not eliminate, formation of 5′-dAH in excess of methylthiolated product.
- Landgraf, Bradley J.,Booker, Squire J.
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supporting information
p. 2889 - 2892
(2016/03/19)
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- Identification and characterization of functional homologs of nitrogenase cofactor biosynthesis protein NifB from methanogens
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Nitrogenase biosynthesis protein NifB catalyzes the radical S-adenosyl-L-methionine (SAM)-dependent insertion of carbide into the M cluster, the cofactor of the molybdenum nitrogenase from Azotobacter vinelandii. Here, we report the identification and characterization of two naturally €truncated€ homologs of NifB from Methanosarcina acetivorans (NifBMa) and Methanobacterium thermoautotrophicum (NifBMt), which contain a SAM-binding domain at the N terminus but lack a domain toward the C terminus that shares homology with NifX, an accessory protein in M cluster biosynthesis. NifBMa and NifBMt are monomeric proteins containing a SAM-binding [Fe4S4] cluster (designated the SAM cluster) and a [Fe4S4]-like cluster pair (designated the K cluster) that can be processed into an [Fe8S9] precursor to the M cluster (designated the L cluster). Further, the K clusters in NifBMa and NifBMt can be converted to L clusters upon addition of SAM, which corresponds to their ability to heterologously donate L clusters to the biosynthetic machinery of A. vinelandii for further maturation into the M clusters. Perhaps even more excitingly, NifBMa and NifBMt can catalyze the removal of methyl group from SAM and the abstraction of hydrogen from this methyl group by 5€-deoxyadenosyl radical that initiates the radical-based incorporation of methyl-derived carbide into the M cluster. The successful identification of NifBMa and NifBMt as functional homologs of NifB not only enabled classification of a new subset of radical SAM methyltransferases that specialize in complex metallocluster assembly, but also provided a new tool for further characterization of the distinctive, NifB-catalyzed methyl transfer and conversion to an iron-bound carbide.
- Fay, Aaron W.,Wiig, Jared A.,Lee, Chi Chung,Hu, Yilin
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p. 14829 - 14833
(2015/12/08)
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- Structural basis of substrate recognition in human nicotinamide N-methyltransferase
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Nicotinamide N-methyltransferase (NNMT) catalyzes the N-methylation of nicotinamide, pyridines, and other analogues using S-adenosyl-l-methionine as donor. NNMT plays a significant role in the regulation of metabolic pathways and is expressed at markedly high levels in several kinds of cancers, presenting it as a potential molecular target for cancer therapy. We have determined the crystal structure of human NNMT as a ternary complex bound to both the demethylated donor S-adenosyl-l-homocysteine and the acceptor substrate nicotinamide, to 2.7 A resolution. These studies reveal the structural basis for nicotinamide binding and highlight several residues in the active site which may play roles in nicotinamide recognition and NNMT catalysis. The functional importance of these residues was probed by mutagenesis. Of three residues near the nicotinamide's amide group, substitution of S201 and S213 had no effect on enzyme activity while replacement of D197 dramatically decreased activity. Substitutions of Y20, whose side chain hydroxyl interacts with both the nicotinamide aromatic ring and AdoHcy carboxylate, also compromised activity. Enzyme kinetics analysis revealed kcat/Km decreases of 2-3 orders of magnitude for the D197A and Y20A mutants, confirming the functional importance of these active site residues. The mutants exhibited substantially increased Km for both NCA and AdoMet and modestly decreased kcat. MD simulations revealed long-range conformational effects which provide an explanation for the large increase in K m(AdoMet) for the D197A mutant, which interacts directly only with nicotinamide in the ternary complex crystal structure.
- Peng, Yi,Sartini, Davide,Pozzi, Valentina,Wilk, Dennis,Emanuelli, Monica,Yee, Vivien C.
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experimental part
p. 7800 - 7808
(2012/05/20)
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- Enzyme-catalyzed transfer of a ketone group from an S-adenosylmethionine analogue: A tool for the functional analysis of methyltransferases
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"Chemical equation presented" S-Adenosylmethionine (AdoMet or SAM)-dependent methyltransferases belong to a large and diverse family of group-transfer enzymes that perform vital biological functions on a host of substrates. Despite the progress in genomics, structural proteomics, and computational biology, functional annotation of methyltransferases remains a challenge. Herein, we report the synthesis and activity of a new AdoMet analogue functionalized with a ketone group. Using catechol O-methyltransferase (COMT, EC 2.1.1.6) and thiopurine S-methyltransferase (TPMT, EC 2.1.1.67) as model enzymes, this robust and readily accessible analogue displays kinetic parameters that are comparable to AdoMet and exhibits multiple turnovers with enzyme. More importantly, this AdoMet surrogate displays the same substrate specificity as the natural methyl donor. Incorporation of the ketone group allows for subsequent modification via bio-orthogonal labeling strategies and sensitive detection of the tagged ketone prod cts. Hence, this AdoMet analogue expands the toolbox available to interrogate the biochemical functions of methyltransferases.
- Lee, Bobby W. K.,Sun, He G.,Zang, Tianzhu,Ju-Kim, Byung,Alfaro, Joshua F.,Zhou, Zhaohui Sunny
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supporting information; experimental part
p. 3642 - 3643
(2010/05/15)
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- Halomethane biosynthesis: Structure of a SAM-dependent halide methyltransferase from arabidopsis thaliana
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It's a gas ! The structure of the halomethane-producing halo/thiocyanate methyltransferase enzyme from plants has been determined. The halide ion and the methyl group of S-adenosyl-L-methionine (SAM) were modeled into the active site (see picture; chloride: green sphere; SAM: C green, O red, S yellow, N blue), which indicated their predisposition for reaction. (Figure Presented)
- Schmidberger, Jason W.,James, Agata B.,Edwards, Robert,Naismith, James H.,O'Hagan, David
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supporting information; experimental part
p. 3646 - 3648
(2010/08/19)
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- Aryltetralin-lignan formation in two different cell suspension cultures of Linum album: Deoxypodophyllotoxin 6-hydroxylase, a key enzyme for the formation of 6-methoxypodophyllotoxin
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Suspension cultures initiated from two different Linum album seedlings accumulate either podophyllotoxin (PTOX, 2.6 mg/g DW) or 6-methoxypodophyllotoxin (6MPTOX, 5.4 mg/g DW) as main lignans. Two molecules of coniferyl alcohol are dimerized to pinoresinol which is converted via several steps into deoxypodophyllotoxin (DOP) which seems to be the branching point to PTOX or 6MPTOX biosynthesis. DOP is hydroxylated at position 7 to give PTOX by deoxypodophyllotoxin 7-hydroxylase (DOP7H). In contrast, 6MPTOX biosynthesis is achieved by DOP hydroxylation at position 6 to β-peltatin by the cytochrome P450 enzyme deoxypodophyllotoxin 6-hydroxylase (DOP6H). The following methylation to β-peltatin-A-methylether is catalyzed by β-peltatin 6-O-methyltransferase (βP6OMT) from which 6MPTOX is formed by hydroxylation at position 7 by β-peltatin-A-methylether 7-hydroxylase (PAM7H). DOP6H and βP6OMT could be characterized in protein extracts from cell cultures of L. flavum and L. nodiflorum, respectively, and here in L. album for the first time. DOP7H and PAM7H activities could not yet be detected with protein extracts. Experiments of feeding DOP together with inhibitors of cytochrome P450 depending as well as dioxygenase enzymes were performed in order to shed light on the type of DOP7H and PAM7H. Growth parameters and specific activities of enzymes from the phenylpropane as well as the lignan specific biosynthetic pathway were measured during a culture period of 16 days. From the enzymes studied only the DOP6H showed a differential activity sustaining the hypothesis that this enzyme is responsible for the differential lignan accumulation in both cell lines.
- Federolf, Katja,Alfermann, A. Wilhelm,Fuss, Elisabeth
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p. 1397 - 1406
(2008/04/12)
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- Rapid conversion of tea catechins to monomethylated products by rat liver cytosolic catechol-O-methyltransferase
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1. The metabolic O-methylation of several catechol-containing tea polyphenols by rat liver cytosolic catechol-O-methyltransferase (COMT) has been studied. 2. When (-)-epicatechin was used as substrate, its O-methylation showed dependence on incubation time, cytosolic protein concentration, incubation pH and concentration of S-adenosyl-L-methionine. The O-methylation of increasing concentrations of (-)-epicatechin followed typical Michaelis-Menten kinetics, and the apparent Km and Vmax were 51 μM and 2882 pmol mg protein-1 min-1, respectively, at pH 7.4, and were 17 μM and 2093 pmol mg protein-1 min-1, respectively, at pH 10.0. 3. Under optimized conditions for in vitro O-methylation, (-)-epicatechin, (+)-epicatechin and (-)-epigallocatechin were rapidly O-methylated by rat liver cytosol. In comparison, (-)-epicatechin gallate and (-)-epigallocatechin gallate were O-methylated at significantly lower rates under the same reaction conditions. 4. COMT-catalysed O-methylation of (-)-epicatechin and (-)-epigallocatechin was inhibited in a concentration-dependent manner by S-adenosyl-L-homocysteine, a demethylated product of S-adenosyl-L-methionine. The IC50 was ca. 10 μM. 5. In summary, the results showed that several catechol-containing tea polyphenols were rapidly O-methylated by rat liver cytosolic COMT. These observations raise the possibility that some of the biological effects of tea polyphenols may be exerted by their O-methylated products or may result from their potential inhibition of the COMT-catalysed O-methylation of endogenous catecholamines and catechol oestrogens.
- Zhu, Bao Ting,Patel, Ushma K.,Cai, May Xiaoxin,Lee, Anthony J.,Conney, Allen H.
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p. 879 - 890
(2007/10/03)
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- Purification and immunological characterization of a recombinant trimethylflavonol 3'-O-methyltransferase
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A flavonol O-methyltransferase cDNA clone (pF3'OMT) from Chrysosplenium americanum was expressed in Escherichia coli Top 10 and the recombinant protein was purified to near homogeneity by affinity chromatography on chelation resin and gel filtration on Superose 12 columns. The purified protein was enzymatically active as a 42 kDa monomer and exhibited strict specificity for position 3' of 3,7,4'-tri methylquercetin. It did not accept the mono- or dimethyl analogs, the parent aglycone quercetin or the phenylpropanoids, caffeic and 5-hydroxyferulic acids as substrates; thus indicating its involvement in the later steps of polymethylated flavonol synthesis in this plant. The K(m) values of the enzyme for 3,7,4'-tri methylquercetin as substrate and S-adenosyl-L-methionine as co-substrate were 7.2 and 20 μM, respectively. The enzyme activity was strongly inhibited by both Ni2+ and p-chloromercuribenzoate and was restored by the addition of EDTA or β-mercaptoethanol, respectively. Antibodies raised against the F3'OMT recombinant protein recognized a protein band migrating at the expected molecular mass of the enzyme on SDS-poly-acrylamide gels of protein extracts prepared from various sources. This implies a high degree of structural similarity among these enzymes that is also corroborated by their hydropathy profiles.
- Seguin, Jacynthe,Muzac, Ingrid,Ibrahim, Ragai K.
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p. 319 - 325
(2007/10/03)
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- PARTIAL PURIFICATION AND SOME PROPERTIES OF AN ALTERNARIOL-O-METHYLTRANSFERASE FROM ALTERNARIA TENUIS
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Key Word Index - Alternaria tenuis; Hyphomycetes; alternariol; coumarin; alternariol-O-methyltransferase; mycotoxin metabolism.A new methyltransferase, alternariol-O-methyltransferase (AOH-MT) was identified in the mould Alternaria tenuis NRRL6434.This enzyme catalyses the methylation of alternariol (AOH), a phenolic mycotoxin, to produce alternariol monomethyl ether (AME).The methyl donor is S-adenosyl-L-methionine (SAM).This activity is cytosolic and is not associated with membrane or particulate material.A 15.5-fold purification was achieved.Experimentation with the partially purified enzyme revealed that AOH-MT is not associated with AOH-synthetase activity.Enzymic activity was associated with a single peak of activity with Mr ca 110 000.This enzyme exhibits optimum activity at pH 8.0.Magnesium stimulates AOH-MT activity.Despite the apparent structural similarity of the substrate to substituted coumarins and cinnamic acids, the cell-free extract displayed no methyltransferase activity towards these compounds.The enzyme has an apparent Km of 38 μM for AOH and an apparent Km of 31 μM for SAM.
- Stinson, E. E.,Moreau, R. A.
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p. 2721 - 2724
(2007/10/02)
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- AN IMPROVED SYNTHESIS OF S-ADENOSYL-L-HOMOCYSTEINE AND RELATED COMPOUNDS
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5'-Chloro-5'-deoxy-2',3'-O-isopropylideneadenosine reacts with disodium salts of L-homocysteine, L-cysteine or 3-mercaptopropanoic acid in liquid ammonia to afford 2',3'-O-isopropylidene derivatives which are easily desalted by chromatography on octadecyl-silica column.On acid treatment, the high purity preparations of S-adenosyl-L-homocysteine, S-adenosyl-L-cysteine, and 5'-carboxyethylthio-5'-deoxyadenosine are obtained in respectable yield.
- Holy, Antonin,Rosenberg, Ivan
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p. 1514 - 1518
(2007/10/02)
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