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。     

豚鼠心脏CDP-乙醇胺磷酸转移酶的动力学研究

为探讨不同的二价基甘油对三种乙醇胺甘油磷脂生物合成能力的影响,通过对豚鼠乙醇胺磷酸转移酶动力学研究,发现磷脂酰乙醇胺的合成可被1-烷基-2-脂酰甘油和1-烯醚基-2-脂酰甘油抑制,而缩醛磷脂酰乙醇胺的生成不受1,2-二脂酰甘油影响,并提示不同的二价基甘油对乙醇胺磷酸转移酶的抑制作用呈非竞争性抑制,此有利于对三种乙醇胺磷脂酰甘油生物合成的相互协调作用。     

盐角草磷酸乙醇胺N-甲基转移酶基因（SePEAMT）启动子的克隆及瞬时表达

磷酸胆碱是合成磷脂酰胆碱和甘氨酸甜菜碱的重要前体,磷酸乙醇胺N-甲基转移酶（PEAMT）是磷酸胆碱合成的关键酶。根据已知的SePEAMT cDNA5'端序列设计两个基因特异的反向引物(PP1,PP2),通过锚定PCR获得了PEAMT起始密码子上游1249bp的序列。RLM-RACE反应确定其转录起始位点A位于起始密码子上游301bp处,由此获得了948bp的SePEAMT启动子序列。PlantCARE和PLACE在线启动子预测工具分析表明：该序列除了含有启动子的基本元件TATA-box和CAAT-box外,还含有一些胁迫诱导元件（如ABRE、HSE、LTR）和花粉特异的激活元件AGAAA。构建了SePEAMT启动子与报告基因GUS 融合的表达载体pPro,并通过农杆菌介导的叶盘法转化烟草,染色结果表明SePEAMT启动子可以有效地驱动GUS基因的瞬时表达。     

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。     

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...     

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...     

大鼠肝CTP:磷酸胆碱胞苷酰转移酶的部分纯化及其特性

 介绍一种部分纯化CTP:磷酸胆碱胞苷酰转移酶(CT)的方法,并对部分纯化CT的性质进行了研究。经盐析、DEAE-纤维素、磷酸-纤维素及CTP-聚琼脂糖柱层析可将大鼠肝胞液中的CT纯化100倍以上,结果重复性好。CT在胞液中及经初步纯化性质稳定,但进一步纯化则其活性很易丧失。鼠肝总磷脂及油酸可激活CT、使其聚合,磷脂酰絲氨酸是其中起主要作用的CT激活物;CT聚合物可被辛基-葡萄糖苷解聚,且保留80％酶活性。用CT常规底物CTP的类似物观察CT的特异性,发现dCTP是比CTP更好的底物,CDP、dCDP能抑制CT对CTP或dCTP的作用,而CMP、dCMP对酶活性无影响。结果提示分子中糖的2′-OH并非CT底物所必需,而和胞苷相连的三个磷酸则不可缺少。     

DNA甲基转移酶

DNA甲基化在基因调节和动物发育中起着重要作用。负责DNA甲基化作用的酶尔为DNA甲基转移酶（Dnmts）。到目前为止,在哺乳动物细胞中已经鉴定了三种DNA甲基转移酶基因家族,即Dnmt1、Dnmt2和Dnmt3。鉴定和研究DNA甲基转移酶对阐明DNA甲基化机制起着关键的作用。     

Phosphoethanolamine N-methyltransferase (PMT-1) catalyses the first reaction of a new pathway for phosphocholine biosynthesis in Caenorhabditis elegans

The development of nematicides targeting parasitic nematodes of animals and plants requires the identification of biochemical targets not found in host organisms. Recent studies suggest that Caenorhabditis elegans synthesizes phosphocholine through the action of PEAMT (S-adenosyl-L-methionine:phosphoethanolamine N-methyltransferases) that convert phosphoethanolamine into phosphocholine. Here, we examine the function of a PEAMT from C. elegans (gene: pmt-1; protein: PMT-1). Our analysis shows that PMT-1 only catalyses the conversion of phosphoethanolamine into phospho-monomethylethanolamine, which is the first step in the PEAMT pathway. This is in contrast with the multifunctional PEAMT from plants and Plasmodium that perform multiple methylations in the pathway using a single enzyme. Initial velocity and product inhibition studies indicate that PMT-1 uses a random sequential kinetic mechanism and is feedback inhibited by phosphocholine. To examine the effect of abrogating PMT-1 activity in C. elegans, RNAi (RNA interference) experiments demonstrate that pmt-1 is required for worm growth and development and validate PMT-1 as a potential target for inhibition. Moreover, providing pathway metabolites downstream of PMT-1 reverses the RNAi phenotype of pmt-1. Because PMT-1 is not found in mammals, is only distantly related to the plant PEAMT and is conserved in multiple parasitic nematodes of humans, animals and crop plants, inhibitors targeting it may prove valuable in human and veterinary medicine and agriculture.     

An Alternative Mechanism for the Methylation of Phosphoethanolamine Catalyzed by Plasmodium falciparum Phosphoethanolamine Methyltransferase

The phosphobase methylation pathway catalyzed by the phosphoethanolamine methyltransferase in Plasmodium falciparum (PfPMT), the malaria parasite, offers an attractive target for anti-parasitic drug development. PfPMT methylates phosphoethanolamine (pEA) to phosphocholine for use in membrane biogenesis. Quantum mechanics and molecular mechanics (QM/MM) calculations tested the proposed reaction mechanism for methylation of pEA involving the previously identified Tyr-19–His-132 dyad, which indicated an energetically unfavorable mechanism. Instead, the QM/MM calculations suggested an alternative mechanism involving Asp-128. The reaction coordinate involves the stepwise transfer of a proton to Asp-128 via a bridging water molecule followed by a typical S_n2-type methyl transfer from S-adenosylmethionine to pEA. Functional analysis of the D128A, D128E, D128Q, and D128N PfPMT mutants shows a loss of activity with pEA but not with the final substrate of the methylation pathway. X-ray crystal structures of the PfPMT-D128A mutant in complex with S-adenosylhomocysteine and either pEA or phosphocholine reveal how mutation of Asp-128 disrupts a hydrogen bond network in the active site. The combined QM/MM, biochemical, and structural studies identify a key role for Asp-128 in the initial step of the phosphobase methylation pathway in Plasmodium and provide molecular insight on the evolution of multiple activities in the active site of the PMT.     

Characterization of glycine sarcosine N-methyltransferase and sarcosine dimethylglycine N-methyltransferase

Glycine betaine is accumulated in cells living in high salt concentrations to balance the osmotic pressure. Glycine sarcosine N-methyltransferase (GSMT) and sarcosine dimethylglycine N-methyltransferase (SDMT) of Ectothiorhodospira halochloris catalyze the threefold methylation of glycine to betaine, with S-adenosylmethionine acting as the methyl group donor. These methyltransferases were expressed in Escherichia coli and purified, and some of their enzymatic properties were characterized. Both enzymes had high substrate specificities and pH optima near the physiological pH. No evidence of cofactors was found. The enzymes showed Michaelis-Menten kinetics for their substrates. The apparent K(m) and V(max) values were determined for all substrates when the other substrate was present in saturating concentrations. Both enzymes were strongly inhibited by the reaction product S-adenosylhomocysteine. Betaine inhibited the methylation reactions only at high concentrations.     

Phosphoethanolamine bases as intermediates in phosphatidylcholine synthesis by lemna

The pathway for synthesis of phosphatidylcholine, the dominant methyl-containing end product formed by Lemna paucicostata, has been investigated. Methyl groups originating in methionine are rapidly utilized by intact plants to methylate phosphoethanolamine successively to the mono-, di-, and tri-methyl (i.e. phosphocholine) phosphoethanolamine derivatives. With continued labeling, radioactivity initially builds up in these compounds, then passes on, accumulating chiefly in phosphatidylcholine (34% of the total radioactivity taken up by plants labeled to isotopic equilibrium with l-[(14)CH(3)]methionine), and in lesser amounts in soluble choline (6%). Radioactivity was detected in mono- and dimethyl derivatives of free ethanolamine or phosphatidylethanolamine only in trace amounts. Pulse-chase experiments with [(14)CH(3)]choline and [(3)H] ethanolamine confirmed that phosphoethanolamine is rapidly methylated and that phosphocholine is converted to phosphatidylcholine. Initial rates indicate that methylation of phosphoethanolamine predominates over methylation of either phosphatidylethanolamine or free ethanolamine at least 99:1. Although more studies are needed, it is suggested this pathway may well turn out to account for most phosphatidylcholine synthesis in higher plants. Phosphomethylethanolamine and phosphodimethylethanolamine are present in low quantities during steady-state growth (18% and 6%, respectively, of the amount of phosphocholine). Radioactivity was not detected in CDP-choline, probably due to the low steady-state concentration of this nucleotide.     

Catalytic mechanism of glycine N-methyltransferase

Methyltransfer reactions are some of the most important reactions in biological systems. Glycine N-methyltransferase (GNMT) catalyzes the S-adenosyl-l-methionine- (SAM-) dependent methylation of glycine to form sarcosine. Unlike most SAM-dependent methyltransferases, GNMT has a relatively high value and is weakly inhibited by the product S-adenosyl-l-homocysteine (SAH). The major role of GNMT is believed to be the regulation of the cellular SAM/SAH ratio, which is thought to play a key role in SAM-dependent methyltransfer reactions. Crystal structures of GNMT complexed with SAM and acetate (a potent competitive inhibitor of Gly) and the R175K mutated enzyme complexed with SAM were determined at 2.8 and 3.0 A resolutions, respectively. With these crystal structures and the previously determined structures of substrate-free enzyme, a catalytic mechanism has been proposed. Structural changes occur in the transitions from the substrate-free to the binary complex and from the binary to the ternary complex. In the ternary complex stage, an alpha-helix in the N-terminus undergoes a major conformational change. As a result, the bound SAM is firmly connected to protein and a "Gly pocket" is created near the bound SAM. The second substrate Gly binds to Arg175 and is brought into the Gly pocket. Five hydrogen bonds connect the Gly in the proximity of the bound SAM and orient the lone pair orbital on the amino nitrogen (N) of Gly toward the donor methyl group (C(E)) of SAM. Thermal motion of the enzyme leads to a collision of the N and C(E) so that a S(N)2 methyltransfer reaction occurs. The proposed mechanism is supported by mutagenesis studies.     

Kinetic mechanism of phosphatidylethanolamine N-methyltransferase

We have investigated the kinetic mechanism of phosphatidylethanolamine (PE) N-methyltransferase purified from rat liver using PE, phosphatidyl-N-monomethylethanolamine (PMME), and phosphatidyl-N,N-dimethylethanolamine (PDME) as substrates. We previously reported (Ridgway, N. D., and Vance, D. E. (1987) J. Biol. Chem. 262, 17231-17239) that initial velocity curves with PE, PMME, and PDME at a fixed concentration of Triton X-100 were sigmoidal, thus generating nonlinear inverse plots. Comparison with other integral membrane enzymes suggested this response resulted from the enzyme's requirement for a complete boundary layer of phospholipid. Hence, the effect of a nonsubstrate phospholipid on initial velocity patterns for PE, PMME, and PDME was examined. The sigmoidicity of initial velocity curves at constant Triton X-100 concentration and increasing PE, PMME, and PDME were converted to the more familiar hyperbolic response by the addition of egg phosphatidylcholine (PC). Hill coefficients for PE, PMME, and PDME at a fixed Triton concentration were 3.6, 2.5, and 4.7, respectively, but with the addition of 30 or 40 mol % of egg PC, coefficients were close to unity (0.9-1.2). The activation by egg PC of PE, PMME, and PDME methylation indicates that a secondary phospholipid binding site(s) plays a role in catalysis in mixed micelles. This site(s) may represent a transmembrane segment(s) in close association with a boundary layer of phospholipid. Kinetic analysis of initial velocity and product inhibition patterns for PMME and PDME methylation fit an ordered Bi Bi mechanism. Phospholipid substrates and products were the first to bind and the last to dissociate from the active site, respectively. As well, PE, PMME, and PDME compete for a single active site. The overall kinetic scheme for the methylation of PE to PC in mixed micelles involves the initial binding of PE, followed by successive steps where S-adenosyl-L-methionine is bound, the sulfonium methyl group is transferred, and S-adenosyl-L-homocysteine is released.