植物甲基化类黄酮及其O-甲基转移酶研究进展

植物类黄酮是重要的药用成分,其生物学功能与化学结构密切相关。O-甲基化修饰可提高类黄酮的稳定性、蛋白亲和力和生物利用度,从而增强其药用活性。O-甲基转移酶(O-methyltransferase)催化类黄酮合成O-甲基化衍生物,是类黄酮代谢途径中的关键修饰酶。本文综述了植物O-甲基化类黄酮的化学结构、药用功能及其药用价值提高机理;并对植物类黄酮O-甲基转移酶的生物学功能、表达调控与开发潜力等进行了总结展望,以期为植物甲基化类黄酮的进一步研究提供新的思路与途径。     

植物磷酸乙醇胺N-甲基转移酶的研究进展

甘氨酸甜菜碱是一种渗透调节物质,能够维持高盐浓度下细胞的渗透平衡和膜的有序性,并有效地稳定酶的结构;胆碱是甘氨酸甜菜碱生物合成的必要前体物质,而磷酸乙醇胺甲基转移酶（phosphoethanolamineN-methyltransferase,PEAMT）作为甲基转移酶,是催化磷酸乙醇胺三次甲基化生成胆碱的限速酶。近年来研究表明磷酸乙醇胺甲基转移酶不仅在植物生长发育过程发挥作用,而且通过参与渗调物质甜菜碱以及胁迫相关第二信使磷脂酸的合成从而使植物对盐胁迫产生应答反应。本文就植物磷酸乙醇胺甲基转移酶的反应作用机理、生物学功能及表达调控机制进行了归纳总结。     

甲基转移酶的功能与分类

甲基转移酶是生物有机体内普遍仔任的一种重要酶类,它能催化遗传物质DNA的甲基化,在基因表达及动物生长、发育中起着重要的调控作用;同时,又能催化多种生理过程中间产物的甲基化进而合成或降解生理活性物质。研究发现,人类的情绪和许多疾病的发生及植物的抗逆性都与甲基转移酶基因的表达有关。     

植物的磷酸乙醇胺甲基转移酶

磷酸乙醇胺甲基转移酶(PEAMT)是催化磷酸乙醇胺(P-EA)甲基化,最终合成磷酸胆碱(P-Cho)的关键酶。文章就近年来植物中PEAMT的结构、性质、功能、表达特性、分子生物学和基因工程的研究进展作了概述。     

DNA甲基化及其对植物发育的调控

DNA甲基化属于一种表观遗传修饰,主要发生在CpG双核苷酸序列中的胞嘧啶上,是在DNA甲基转移酶催化下,以S-腺苷甲硫氨酸为甲基供体,将甲基转移到胞嘧啶上,生成5-甲基胞嘧啶的一种反应。DNA甲基化在植物生长过程中具有极其重要的作用。综述了植物DNA甲基化的特征、调控机制,及其对植物基因表达影响的研究进展。     

植物SABATH甲基转移酶家族生物学功能研究进展

植物SABATH甲基转移酶是甲基转移酶家族中重要的一类,该家族以最先发现的3个家族成员的基因名称缩写命名,是一类能够催化植物激素和小分子化合物的羧基和N原子甲基化的酶,在植物激素及相关信号分子代谢中行使了重要的生物学功能。研究表明,SABATH甲基转移酶不仅直接参与调控植物激素代谢、挥发性化合物的合成,还能通过改变植物次生代谢影响有益和有害昆虫的行为,从而帮助植物完成生命周期,保护植物免受病虫危害。本文简要综述SABATH甲基转移酶的分类及其在植物生长、病虫害防御反应等生物过程中的作用的研究进展。     

植物SABATH甲基转移酶研究进展

近年来对植物甲基转移酶（methyltransferases,MTs）的研究发现了新一类成员,并用最初发现的3个酶将其命名为SA-BATH甲基转移酶（SABATHMTs）,这3个酶分别是水杨酸羧基甲基转移酶（salicylic acid carboxyl methyltransferases,SAMT）、苯甲酸羧基甲基转移酶（benzoic acid carboxyl methyltransferases,BAMT）和可可碱合酶（theobromine synthase）。SABATHMTs能对植物激素和其他一些小分子物质进行N位或O位甲基化形成相应的甲基化产物,在植物次生代谢、发育及防御中起重要作用。本文从SABATHMTs潜在底物、进化及调控等方面综述了近年来对该家族的研究。     

DNA甲基转移酶的表达调控及主要生物学功能

DNA甲基化是表观遗传学的重要部分, 同组蛋白修饰相互作用, 通过改变染色质结构, 调控基因表达。在哺乳类细胞或人体细胞中, DNA甲基化与细胞的增殖、衰老、癌变等生命现象有着重大关系。对催化DNA甲基化的DNA甲基转移酶(DNA methyltransferase, Dnmt)的研究可以揭示DNA甲基化对基因表达调控的机制, 从而研究与之相关的重要生命活动。文章以DNA甲基转移酶作为切入点, 探讨DNA甲基转移酶在基因表达调控中发挥的作用及其主要生物学功能。     

PRMT5在癌症中的研究进展

蛋白质精氨酸甲基转移酶(protein arginine methyltransferases,PRMTs)是真核生物中常见的一种酶,可催化组蛋白和非组蛋白底物中的精氨酸残基发生甲基化.在人类的基因组中,PRMTs由9个基因编码.作为最主要的Ⅱ型精氨酸甲基转移酶,PRMT5是PRMT家族成员之一,参与了包括信号转导、转...     

DNA甲基化与植物的生长发育

文章就DNA甲基化与植物生长发育的关系、催化甲基化的酶、5－甲基胞嘧啶在植物基因组中的分布、甲基化的发生和遗传的研究进展作了介绍。     

Regioselectivity of 7-O-methyltransferase of poplar to flavones

POMT-7, an O-methyltransferase from poplar (Populus deltoids) was used to modify a variety of flavonoid compounds. POMT-7 was able to transfer a methyl group to several flavonoids containing a C-7 hydroxyl group. However, POMT-7 showed a higher affinity toward flavonol and flavone such as apigenin, kaempferol, luteolin, and quercetin than flavanone and isoflavone. Based on comparison of HPLC retention times with authentic compounds and corresponding nuclear magnetic resonance spectroscopy data, the methylation position of the reaction products was determined to be at the hydroxyl group of C-7. Biotransformation kinetics indicated that the enzyme converted more than 80% of the apigenin, kaempferol, luteolin and quercetin substrates, which were added at concentration of 70 microM, into corresponding 7-methoxy compounds within 24 h.     

Regioselectivity of catechol O-methyltransferase. The effect of pH on the site of O-methylation of fluorinated norepinephrines

Selectivity of catechol O-methyltransferase has been examined for the three ring-fluorinated norepinephrines to elucidate the role of acidity of the phenolic groups in their methylation. Substitution of fluorine at the 5-position of norepinephrine reverses the selectivity of catechol O-methyltransferase so that p-O-methylation predominates. The 5-fluoro substituent also causes the pKa of the p-hydroxyl group to decrease substantially. In contrast, 2- and 6-fluoronorepinephrines are methylated predominantly at the m-hydroxyl group. These results suggest that acidity of a phenolic group can play an important role in its ability to be methylated by catechol O-methyltransferase. Percentages of p-O-methylation of norepinephrine and its fluorinated derivatives increase with pH. This relative increase in p-O-methylation appears to accompany ionization of a group with pKa of 8.6, 7.7, 7.9, and 8.4 for norepinephrine and its 2-, 5-, and 6-fluoro derivative, respectively. These pKa values are the same as or similar to the pKa values of a phenolic hydroxyl group of these substrates. 3,4-Dihydroxybenzyl alcohol and its 5-fluoro derivative are O-methylated by catechol O-methyltransferase to form p- and m-O-methyl products in approximately 1:1 and 4:1 ratios, respectively, at all pH values. Based on the above results, a catechol-binding site model for catechol O-methyltransferase is proposed in which the two phenolic hydroxyl groups of catechol substrates are postulated to be approximately equally spaced from the methyl group of the cosubstrate S-adenosylmethionine.     

Inhibition of LDL oxidation by flavonoids in relation to their structure and calculated enthalpy

Twenty flavonoid compounds of five different subclasses were selected, and the relationship of their structure to the inhibition of low-density lipoprotein (LDL) oxidation in vitro was investigated. The most effective inhibitors, by either copper ion or 2,2'-azobis (2-amidino-propane) dihydrochloride (AAPH) induction, were flavonols and/or flavonoids with two adjacent hydroxyl groups at ring B. In the presence of the later catechol group, the contribution of the double bond and the carbonyl group at ring C was negligible. Isoflavonoids were more effective inhibitors than other flavonoid subclasses with similar structure. Substituting ring B with hydroxyl group(s) at 2' position resulted in a significantly higher inhibitory effect than by substituting ring A or ring B at other positions. The type of LDL inducer had no effect in flavonoids with catechol structure. Calculated heat of formation data (deltadeltaH(f)) revealed that the donation of a hydrogen atom from position 3 was the most likely result, followed by that of a hydroxyl from ring B. Position 3 was favored only in the presence of conjugated double bonds between ring A to ring B. This study makes it possible to assign the contribution of different functional groups among the flavonoid subclasses to in vitro inhibition of LDL oxidation.     

Role of Serine 286 in cosubstrate binding and catalysis of a flavonol O-methyltransferase.

O-Methyltransferases catalyze the transfer of the methyl groups of S-adenosyl-L-methionine to specific hydroxyl groups of several classes of flavonoid compounds. Of the several cDNA clones isolated from a Chrysosplenium americanum library, FOMT3' encodes the 3'/5'-O-methylation of partially methylated flavonols. The recombinant protein of another clone, FOMTx which differs from FOMT3' by a single amino acid residue (Ser286Arg) exhibits no enzymatic activity towards any of the flavonoid substrates tested. Replacement of Ser 286 in FOMT3' with either Ala, Leu, Lys or Thr, almost abolished O-methyltransferase activity. In contrast with FOMT3', no photoaffinity labeling could be achieved using [(14)CH(3)]AdoMet with the mutant recombinant proteins indicating that Ser 286 is also required for cosubstrate binding. These results are corroborated by isothermal titration microcalorimetry measurements. Circular dichroism spectra ruled out any significant conformational differences in the secondary structures of both FOMT3' and Ser286Arg. Modeling FOMT3' on the structure of chalcone methyltransferase indicates that serine 286 is greater than 10 A from any of the residues of the active site or the AdoMet binding site of FOMT3'. At the same time, residues 282 to 290 are conserved in most of the Chrysosplenium americanum OMTs. These residues form a large part of the subunit interface, and at least five of these residues are within 4 A of the opposing subunit. It would appear, therefore, that mutations in Ser286 exert their influence by altering the contacts between the subunits and that these contacts are necessary for maintaining the integrety of the AdoMet binding site and active site of this group of enzymes.     

Characterization of an O-methyltransferase from soybean.

O-methyltransferases (OMTs) catalyze the transfer of a methyl group from S-adenosine-L-methionine to a hydroxyl group of an acceptor molecule to form methyl ether derivatives and can modify the basic backbone of a secondary metabolite. A new O-methyltransferase, SOMT-9, was cloned from Glycine max and found to encode a protein whose molecular weight is 27-kDa. SOMT-9 was expressed as a GST-fusion protein in Escherichia coli and several compounds such as caffeic acid, esculetin, narigenin, kaempferol, quercetin, and luteolin were tested as putative substrates of SOMT-9. HPLC and NMR results showed that SOMT-9 transfers a methyl group to the 3'-OH group of substrates having ortho-hydroxyl groups. SOMT-9 showed the highest affinity for quercetin, suggesting that SOMT-9 uses a flavonoid as a substrate. Based on its molecular weight and substrate specificity, SOMT-9 belongs to a new class of OMT and is likely to be involved in the biosynthesis of isorhamnetin.     

Purification and characterization of S-adenosyl-L-methionine: caffeic acid 3-O-methyltransferase from suspension cultures of alfalfa (Medicago sativa L.)

Caffeic acid O-methyltransferase (COMT) is one of a group of proteins present in alfalfa cell cultures which can be photoaffinity labeled with S-adenosyl-L-[methyl-3H]methionine. The enzyme was purified to homogeneity from elicitor-treated suspension cultures and shown to exist as an active monomer of subunit Mr 41,000. COMT could be separated into two forms on the basis of their isoelectric points and relative affinities for S-adenosyl-methionine and S-adenosylhomocysteine. Both forms had equal affinities for caffeic acid, were highly specific for the 3-hydroxyl group of substituted cinnamic acids, and exhibited negligible activity toward flavonoid substrates. An antiserum raised against COMT from aspen immunoprecipitated alfalfa COMT activity. Peptide mapping studies indicated that the two forms of COMT and an isoflavone O-methyltransferase from alfalfa are closely related proteins. The extractable activity of COMT doubled over a 48-h period following exposure of alfalfa cell suspensions to a yeast elicitor preparation, and this was associated with a small change in the relative proportions of the two forms of the enzyme.     

The non-additive contribution of hydroxyl substituents to Akt kinase–apigenin affinity

The natural product apigenin is a flavonoid derivative substituted by three hydroxyl functional groups at positions 4′, 5 and 7 [OH(4′), OH(5) and OH(7)] of the basic flavonoid skeleton, which has shown strong inhibition on the development, proliferation and invasion of tumour cells by binding specifically to Akt kinase to inactivate the Akt signalling pathway. In this study, a typical non-additivity of the three hydroxyl substituents’ contributions to Akt–apigenin binding affinity is demonstrated by combination of four empirical scoring functions, molecular dynamics simulations, molecular mechanics-Poisson–Boltzmann/surface area (MM/PBSA) analyses and hybrid quantum mechanics/molecular mechanics (QM/MM) calculations. It is found that (i) the empirical scoring functions are incapable of properly reflecting the non-additivity feature, which, however, can be well described by the more rigorous MM/PBSA and QM/MM methods, (ii) the hydroxyl group contributions to ligand binding affinity are deviated significantly from linear additive model due to the strong conjugate effect and σ-effect among them, that is, the co-contribution of the three hydroxyl groups is far less than the sum of their individual contributions and (iii) as might be expected, a strong interactive effect is observed for the two adjacent substituents OH(5) and OH(7) as compared with that of distant OH(5) and OH(4′) as well as OH(7) and OH(4′). In addition, the structural basis, energetic property and molecular mechanism of the non-additivity feature are also explored in detail using the natural population analysis and quantum mechanical calculations.     

Regiospecificity and kinetic properties of a plant natural product O-methyltransferase are determined by its N-terminal domain

A recently discovered, S-adenosyl-L-methionine and bivalent cation-dependent O-methyltransferase from the ice plant, Mesembryanthemum crystallinum, is involved in the methylation of various flavonoid and phenylpropanoid conjugates. Differences in regiospecificity as well as altered kinetic properties of the recombinant as compared to the native plant O-methyltransferase can be attributed to differences in the N-terminal part of the protein. Upon cleavage of the first 11 amino acids, the recombinant protein displays essentially the same substrate specificity as observed earlier for the native plant enzyme. Product formation of the newly designed, truncated recombinant enzyme is consistent with light-induced accumulation of methylated flavonoid conjugates in the ice plant. Therefore, substrate affinity and regiospecificity of an O-methyltransferase in vivo and in vitro can be controlled by cleavage of an N-terminal domain.     

Purification, characterization, and kinetic mechanism of S-adenosyl-L-methionine: vitexin 2"-O-rhamnoside 7-O-methyltransferase of Avena sativa L

An O-methyltransferase catalyzing the transfer of the methyl group of S-adenosyl-L-methionine to the A-ring 7-hydroxyl group of vitexin 2"-O-rhamnoside has been isolated from oat primary leaves and purified 180-fold by protein fractionation with (NH4)2SO4 and chromatography on DEAE-cellulose and S-adenosyl-L-homocysteine-sepharose. Km values for S-adenosyl-L-methionine and the flavonoid substrate were 1.6 microM and 15 microM, respectively. The lack of methyltransfer to biosynthetic intermediates suggests that the reaction is the last step in the biosynthetic pathway to the oat flavonoid 7-O-methylvitexin 2"-O-rhamnoside. Based on results obtained from kinetic inhibition studies and affinity chromatography a mono-iso Theorell-Chance mechanism is proposed with the nucleotide substrate binding before the flavonoid.     

Sequential O-methylation of tricetin by a single gene product in wheat

Flavonoid compounds are ubiquitous in nature. They constitute an important part of the human diet and act as active principles of many medicinal plants. Their O-methylation increases their lipophilicity and hence, their compartmentation and functional diversity. We have isolated and characterized a full-length flavonoid O-methyltransferase cDNA (TaOMT2) from a wheat leaf cDNA library. The recombinant TaOMT2 protein was purified to near homogeneity and tested for its substrate preference against a number of phenolic compounds. Enzyme assays and kinetic analyses indicate that TaOMT2 exhibits a pronounced preference for the flavone, tricetin and gives rise to three methylated enzyme reaction products that were identified by TLC, HPLC and ESI-MS/MS as its mono-, di- and trimethyl ether derivatives. The sequential order of tricetin methylation by TaOMT2 is envisaged to proceed via its 3'-mono--->3',5'-di--->3',4',5'-trimethyl ether derivatives. To our knowledge, this is the first report of a gene product that catalyzes three sequential O-methylations of a flavonoid substrate.