molecules Article The Functional Characterization of a Site-Specific Apigenin 40-O-methyltransferase Synthesized by the Liverwort Species Plagiochasma appendiculatum Hui Liu, Rui-Xue Xu, Shuai Gao and Ai-Xia Cheng * Key Laboratory of Chemical Biology of Natural Products, Ministry of Education, School of Pharmaceutical Sciences, Shandong University, Jinan 250012, China; [email protected] (H.L.); [email protected] (R.-X.X.); [email protected] (S.G.) * Correspondence: [email protected]; Tel.: +86-531-8838-2012; Fax: +86-531-8838-2019 Academic Editors: Qing-Wen Zhang and Chuangchuang Li Received: 5 April 2017; Accepted: 4 May 2017; Published: 7 May 2017 Abstract: Apigenin, a widely distributed flavone, exhibits excellent antioxidant, anti-inflammatory, and antitumor properties. In addition, the methylation of apigenin is generally considered to result in better absorption and greatly increased bioavailability. Here, four putative Class II methyltransferase genes were identified from the transcriptome sequences generated from the liverwort species Plagiochasma appendiculatum. Each was heterologously expressed as a His-fusion protein in Escherichia coli and their methylation activity against apigenin was tested. One of the four Class II OMT enzymes named 40-O-methyltransferase (Pa40OMT) was shown to react effectively with apigenin, catalyzing its conversion to acacetin. Besides the favorite substrate apigenin, the recombinant PaF40OMT was shown to catalyze luteolin, naringenin, kaempferol, quercetin, genistein, scutellarein, and genkwanin to the corresponding 40-methylation products. In vivo feeding experiments indicated that PaF40OMT could convert apigenin to acacetin efficiently in E. coli and approximately 88.8 µM (25.2 mg/L) of product was synthesized when 100 µM of apigenin was supplemented. This is the first time that a Class II plant O-methyltransferase has been characterized in liverworts. Keywords: Plagiochasma appendiculatum; Class II O-methyltransferase; apigenin; acacetin; feeding 1. Introduction Flavonoids are a family composed of various polyphenolic compounds synthesized by plant species. Among this family, apigenin (4,5,7-trihydroxyflavone) is a widely distributed flavone and exhibits antioxidant, anti-inflammatory, and antitumor properties [1–4]. It was found that methylation of the free hydroxy groups in the flavonoids dramatically increases their stability and enhances the membrane transport, resulting in better absorption and greatly increased bioavailability [5,6]. Acacetin, 40-methylated form of apigenin, has been proven to have broad-spectrum biological activities and has gradually been used in the treatment of multifarious diseases. A previous study revealed that acacetin is a promising atrium-selective agent for the treatment of anti-atrial fibrillation [7] and has been well documented to show novel pharmacological properties such as antinociceptive, anti-inflammatory, and anti-cancer activities [8–10]. Enzyme-catalyzed synthesis of acacetin can be achieved by the para-O-methylation of apigenin using S-adenosyl-L-methionine as a methyl donor catalyzed by O-methyltransferases (OMTs). O-methyltransferases are known to be involved in the reaction transforming methyl to hydroxy groups of phenylpropanoids and flavonoids. Based on their size, amino acid sequence, and cation dependency, they have been classified into two major groups, referred to as Classes I and II. Class IOMTs (caffeoyl-coenzyme A O-methyltransferases; CCoAOMT) Molecules 2017, 22, 759; doi:10.3390/molecules22050759 www.mdpi.com/journal/molecules Molecules 2017, 22, 759 2 of 11 Molecules 2017, 22, 759 2 of 11 KDa methylate the lignin precursors caffeoyl-coenzyme A (CCoA) and 5-hydroxyferuloyl CoA in the presence of an Mg2+ cation premise. Substrates preferred by CCoAOMT possess two vicinal hydroxylwith a molecular groups weightand their of 26–29 methylation KDa methylate affects thethe ligninmeta- precursorshydroxyl group. caffeoyl-coenzyme COMTs (caffeic A (CCoA) acid 2+ Oand-methyltransferases) 5-hydroxyferuloyl are CoA cation-independent in the presence enzymes of an Mg capablecation of methylating premise. Substratesnot only flavonoids, preferred but by - alsoCCoAOMT both 3-hydroxyl-and possess two vicinal 5-hydroxyl-containing hydroxyl groups phenylpropanoid-derived and their methylation affects lignin the precursors meta hydroxyl [11]. group.Class O IICOMTs OMTs’ (caffeic molecular acid weights-methyltransferases) range from are38 cation-independentto 43 KDa, and their enzymes activity capable does of not methylating require the not presenceonly flavonoids, of a metal but alsocation. both Beside 3-hydroxyl-ands the typical 5-hydroxyl-containing COMT activity of catalyzing phenylpropanoid-derived the methylation of lignin the ligninprecursors precursors [11]. Class caffeic II OMTs’ acid and molecular 5-hydroxy weights coniferic range acid, from some 38 to 43Class KDa, II andOMTs their also activity play a does role not in therequire methylation the presence of flavonoids. of a metal cation. For instance, Besides thethe typicalArabidopsis COMT thaliana activityenzyme of catalyzing AtCOMT1 the methylation is able to methylateof the lignin the precursors flavonol quercetin, caffeic acid which and 5-hydroxy bears an orthodihydroxy coniferic acid, some group, Class while II OMTs the Populus also play deltoides a role enzymein the methylation POMT-7 transfers of flavonoids. a methyl For instance,group to the flavonoidsArabidopsis (apigenin, thalianaenzyme kaempferol, AtCOMT1 luteolin, is able and to quercetin)methylate to the the flavonol C-7 OH quercetin, group [12]. which bears an orthodihydroxy group, while the Populus deltoides enzymeIn recent POMT-7 years, transfers metabolic a methyl engineering group to containing flavonoids OMT (apigenin, genes kaempferol, has been one luteolin, of theand hottest quercetin) fields into theterms C-7 of OH use group for the [12 ].production of valuable medical compounds, especially for polyhydroxyl molecules.In recent O-methylation years, metabolic of a engineering specific OH containing group th OMTrough genes chemical has been synthesis one of theconfronts hottest many fields drawbacks:in terms of harsh use for catalytic the production conditions, of valuablea long reac medicaltion time, compounds, protection especially of unwanted for polyhydroxyl groups, and expensivemolecules. methylationO-methylation reagents. of a For specific the production OH group of through acacetin, chemical direct C-4 synthesis′ methylation confronts of apigenin many isdrawbacks: impossible harsh by chemical catalytic means, conditions, due ato long the reactionhigher acidity time, protectionof C-7 hydroxy of unwanted group. groups,It has been and 0 synthesizedexpensive methylation from largely reagents. available For naringin the production via dehydrogenation of acacetin, direct (to C-4 rhoifolin)methylation and hydrolysis of apigenin of is glycosidicimpossible side by chemical chain [13,14]. means, However, due to the microbial higher acidity biotransformation of C-7 hydroxy is group. an alternative It has been strategy synthesized with greatfrom largelypotential available to produce naringin novel via bioactive dehydrogenation compounds (to in rhoifolin) a one-step and reaction hydrolysis and of a glycosidic much milder side reactionchain [13 condition.,14]. However, Microbes microbial have biotransformation a highly tractable is genetic an alternative system strategyand favorable with great fermentation potential conditionsto produce [15]. novel Several bioactive studies compounds have shown in a that one-step high reactionproductivities and a of much O-methylation milder reaction for flavonoids condition. areMicrobes viable. have SaOMT-2, a highly isolated tractable from genetic Streptomyces system and avermitilis favorable fermentationMA-4680, converted conditions naringenin [15]. Several to sakuranetinstudies have in shown E. coli, thatwhich high has productivities an antifungal ofactivityO-methylation against Magnaporthe for flavonoids grisea are [16]. viable. SaOMT-2, isolatedLiverworts from Streptomyces produce methylated avermitilis MA-4680,flavonoids, converted lignans, lignin-like naringenin compound to sakuranetins, and in bis-bibenzyls.E. coli, which Here,has an a antifungaldescription activity is given against of theMagnaporthe isolation of griseafour Plagiochasma[16]. appendiculatum Class II OMTs, one of whichLiverworts has been produce functionally methylated characterized flavonoids, as lignans,a flavone lignin-like 4′-O-methyltransferase compounds, and (Pa4 bis-bibenzyls.′OMT) with apigeninHere, a description as the favorite is given substrate of the isolation(Figure of1). fourThePlagiochasma site-specific appendiculatumbiotransformationClass of II apigenin OMTs, one to 0 0 acacetinof which in has engineered been functionally E. coli expressing characterized PaF4′ asOMT a flavone was explored 4 -O-methyltransferase and approximately (Pa4 25.2OMT) mg/L with of acacetinapigenin was as the synthesized. favorite substrate This is (Figure the first1). Thetime site-specific that a Class biotransformation II plant O-methyltransfe of apigeninrase, to acacetin which 0 catalyzedin engineered the E.O-methylation coli expressing of PaF4apigeninOMT to was form explored acacetin and in approximately E. coli, has been 25.2 mg/Lcharacterized of acacetin in liverworts.was synthesized. This is the first time that a Class II plant O-methyltransferase, which catalyzed the O-methylation of apigenin to form acacetin in E. coli, has been