979-92-0Relevant articles and documents
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
Grove, Tyler L.,Radle, Matthew I.,Krebs, Carsten,Booker, Squire J.
, p. 19586 - 19589 (2011)
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.
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Cantoni,Scarano
, p. 4744 (1954)
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S-adenosyl-L-methionine: Anol-O-methyltransferase activity in organ cultures of Pimpinella anisum
Kemmerer, Birgitt,Reichling, Juergen
, p. 397 - 403 (1996)
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.
Expression and purification of an ArsM-elastin-like polypeptide fusion and its enzymatic properties
Ke, Changdong,Xiong, Hui,Zhao, Chungui,Zhang, Zhigang,Zhao, Xiaolan,Rensing, Christopher,Zhang, Guangya,Yang, Suping
, p. 2809 - 2820 (2019)
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.
Three-Dimensional Proteome-Wide Scale Screening for the 5-Alpha Reductase Inhibitor Finasteride: Identification of a Novel Off-Target
Giatti, Silvia,Di Domizio, Alessandro,Diviccaro, Silvia,Falvo, Eva,Caruso, Donatella,Contini, Alessandro,Melcangi, Roberto Cosimo
, p. 4553 - 4566 (2021/05/06)
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.
Biosynthesis of methyl (E)-cinnamate in the liverwort Conocephalum salebrosum and evolution of cinnamic acid methyltransferase
Zhang, Chi,Chen, Xinlu,Crandall-Stotler, Barbara,Qian, Ping,K?llner, Tobias G.,Guo, Hong,Chen, Feng
, p. 50 - 59 (2019/05/14)
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.