Systemically functional characterization of regiospecific flavonoid O-methyltransferases from Glycine max

Plants produce diverse flavonoids for defense and stress resistance,most of which have health benefits and are widely used as food additives and medicines.Methylation of the free hydroxyl groups of flavonoids,catalyzed by S-adenosyl-l-methionine-dependent O-methyltransferases(OMTs),significantly affects their physicochemical properties and bioactivities.Soybeans(Glycine max)contain a rich pool of O-methylated flavonoids.However,the OMTs responsible for flavonoid methylation in G.max remain largely unknown.We screened the G.max genome and obtained 22 putative OMT-encoding genes that share a broad spectrum of amino acid identities(25–96%);among them,19 OMTs were successfully cloned and heterologously expressed in Escherichia coli.We used the flavonoids containing the free 3,5,7,8,3′,4′hydroxyl group,such as flavones(luteolin and 7,8-dihydroxyflavone),flavonols(kaempferol and quercetin),flavanones(naringenin and eriodictyol),isoflavonoids(daidzein and glycetein),and caffeic acid as substrates,and 15 OMTs were proven to catalyze at least one substrate.The methylation activities of these GmOMTs covered the 3,7,8,3′,4′-hydroxyl of flavonoids and 7,4′-hydroxyl of isoflavonoids.The systematic characterization of G.max flavonoid OMTs provides insights into the biosynthesis of methylated flavonoids in soybeans and OMT bioparts for the production of methylated flavonoids via synthetic biology.

Natural sources,refined extraction,biosynthesis,metabolism,and bioactivities of dietary polymethoxyflavones(PMFs)

Polymethoxyflavones(PMFs)are a type of uncommon dietary flavonoids,characterized by more than one methoxy group,which exist in limited plant species,like Citrus species and Kaempferia parviflora.In addition,different PMFs,such as nobiletin,sinensetin,tangeretin,and casticin,have been isolated from these natural sources.PMFs have received increasing attention due to their multiple bioactivities,such as antioxidant,anti-inflammatory,anti-cancer,metabolic regulatory,immunoregulatory,neuroprotective,and skin protective effects.These bioactivities of PMFs should be associated with the regulation of critical molecular targets and the interaction with gut microbiota.In order to provide a comprehensive and updated review of PMFs,their natural sources,refined extraction,biosynthesis,metabolism,and bioactivities are summarised and discussed,with the emphasis on the molecular mechanisms of PMFs on regulating different chronic diseases.Overall,PMFs may be promising flavonoids to the forefront of nutraceuticals for the prevention and/or treatment of certain human chronic diseases.

O-methyltransferase CbzMT catalyzes iterative 3,4-dimethylations for carbazomycin biosynthesis

Carbazomycins(1-8)are a subgroup of carbazole derivatives that contain oxygen at the C3 and C4 positions and an unusual asymmetric substitution pattern.Several of these compounds exhibit antifungal and antioxidant activities.To date,no systematic biosynthetic studies have been conducted on carbazomycins.In this study,carbazomycins A and B(1 and 2)were isolated from Streptomyces luteosporeus NRRL 2401 using a one-strain-many-compound(OSMAC)-guided natural product mining screen.A biosynthetic gene cluster(BGC)was iden-tified,and possible biosynthetic pathways for 1 and 2 were proposed.The in vivo genetic manipulation of the O-methyltransferase-encoding gene cbzMT proved indispensable for 1 and 2 biosynthesis.Size exclusion chro-matography indicated that CbzMT was active as a dimer.In vitro biochemical assays confirmed that CbzMT could repeatedly act on the hydroxyl groups at C3 and C4,producing monomethylated 2 and dimethylated 1.Monomethylated carbazomycin B(2)is not easily methylated;however,CbzMT seemingly prefers the dimethy-lation of the dihydroxyl substrate(12)to 1,even with a low conversion efficiency.These findings not only improve the understanding of carbazomycin biosynthesis but also expand the inventory of OMT-catalyzing it-erative methylations on different acceptor sites,paving the way for engineering biocatalysts to synthesize new active carbazomycin derivatives.

Revealing the role of CCoAOMT1: fine-tuning bHLH transcription factors for optimal anther development

The tapetum,a crucial innermost layer encompassing male reproductive cells within the anther wall,plays a pivotal role in normal pollen development.The transcription factors (TFs) bHLH010/089/091 redundantly facilitate the rapid nuclear accumulation of DYSFUNCTIONAL TAPETUM 1,a gatekeeper TF in the tapetum.Nevertheless,the regulatory mechanisms governing the activity of bHLH010/089/091 remain unknown.In this study,we reveal that caffeoyl coenzyme A O-methyltransferase 1 (CCoAOMT1) is a negative regulator affecting the nuclear localization and function of bHLH010 and bHLH089,probably through their K259 site.Our findings underscore that CCoAOMT1 promotes the nuclear export and degradation of bHLH010 and bHLH089.Intriguingly,elevated CCoAOMT1 expression resulted in defective pollen development,mirroring the phenotype observed in bhlh010 bhlh089 mutants.Moreover,our investigation revealed that the K259A mutation in the bHLH089 protein disrupted its translocation from the nucleus to the cytosol and impeded its degradation induced by CCoAOMT1.Importantly,transgenic plants with the probHLH089::bHLH089^(K259A)construct failed to rescue proper pollen development or gene expression in bhlh010 bhlh089 mutants.Collectively,these findings emphasize the need to maintain balanced TF homeostasis for male fertility.They firmly establish CCoAOMT1 as a pivotal regulator that is instrumental in achieving equilibrium between the induction of the tapetum transcriptional network and ensuring appropriate anther development.

Cloning of a caffeoyl-coenzyme A O-methyltransferase from Camellia sinensis and analysis of its catalytic activity

Epigallocatechin-3-O-（3-O-methyl） gallate（EGCG3＂Me） present in leaves of Camellia sinensis has many beneficial biological activities for human health. However, EGCG3＂Me occurs naturally in tea leaves in extremely limited quantities. Finding an enzyme from C. sinensis to catalyze the synthesis of EGCG3＂Me is an alternative method to make up for the scarcity of EGCG3＂Me in natural situations. In the present study, a complementary DNA（c DNA） encoding region and genomic DNA of the caffeoyl-coenzyme A O-methyltransferase（CCo AOMT） gene were isolated from C. sinensis（designated Cs CCo AOMT）. Nucleotide sequence analysis of Cs CCo AOMT revealed an open reading frame of 738 bp that encodes a polypeptide with a predicted molecular weight of 28 k Da, which correlated well with the results of sodium dodecyl sulfate-polyacrylamide gel electrophoresis（SDS-PAGE）. The full-length DNA sequence（2678 bp） contained five exons and four introns. The deduced amino acid sequence of Cs CCo AOMT shared 92% identity with CCo AOMTs from Codonopsis lanceolata and Betula luminifera. The catalytic activity of Cs CCo AOMT was analyzed. Three monomethylated epigallocatechin-3-O-gallate（EGCG） compounds（EGCG4＂Me, EGCG3＂Me, and EGCG3＇Me） were produced by Cs CCo AOMT with K m in the micromolar range. Real-time polymerase chain reaction（RT-PCR） experiments indicated that the Cs CCo AOMT transcript was present at low levels during the early stages of leaf maturity（the first leaf and bud on a shoot） but the relative expression was augmented at advanced stages of leaf maturity（the third or fourth leaf on a shoot）, which accorded well with changes in EGCG3＂Me content in fresh leaves. Hence, we concluded that Cs CCo AOMT catalyzes the syntheses of methylated EGCGs.

Functional characterization of caffeic acid O-methyltransferase in internode lignification of switchgrass(Panicum virgatum)

Caffeic acid O-methyltransferase(COMT) is a crucial enzyme that mainly methylates phenylpropanoid meta-hydroxyl of C5 in the biosynthesis of syringyl lignin in angiosperms. A putative COMT, named as PvCOMT1,was isolated from switchgrass(Panicum virgatum), a C4 warm-season dual-purpose forage and bioenergy crop. Our results showed that recombinant PvCOMT1 enzyme protein catalyzed the methylation of 5-OH coniferyl alcohol, 5-OH coniferaldehyde(CAld5H) and 5-OH ferulic acid. Further in vitro studies indicate that CAld5H can dominate COMT-mediated reactions by inhibiting the methylation of the other substrates. Transgenic switchgrass plants generated by an RNAi approach were further employed to study the function of COMT in internode lignification. A dramatic decrease in syringyl lignin units coupled with an obvious incorporation in 5-OH guaiacyl lignin units were observed in the COMT-RNAi transgenic plants. However, the constitutive suppression of COMT in switchgrass plants altered neither the pattern of lignin deposition along the stem nor the anatomical structure of internodes. Consistent with the biochemical characterization of PvCOMT1, a significant decrease in sinapaldehyde was found in the COMT-RNAi transgenic switchgrass plants, suggesting that CAld5H could be the optimal intermediate in the biosynthesis syringyl lignin.

Methylation in hangtaimycin biosynthesis and its antibacterial activities

About two-thirds of small molecule drugs contain methyl group and it plays a very important role in the drug development.So,methyltransferases catalyzing the methylation have always attracted great attention.Hangtaimycin(HTM)is a potent hepatoprotective agent.Previous study showed that its biosynthetic gene cluster contained three methyltransferase domains,but their characteristics in HTM biosynthetic pathway has not been revealed.In this study,we clarified multi-methylations in HTM biosynthesis in vivo.It showed that the two S-adenosylmethionine-dependent methyltransferases(SAM-MTs)of HtmA2(-module 6)-MT domain and HtmB2(-module 18)-MT domain are responsible for the installation of methyl group at C-45 and N-12,respectively,whereas the FK506 methyltransferase(FKMT)type O-methyltransferase of HtmB1(-module 16)-MT domain take care of the methylation at O-21 of HTM.We also reported the antibacterial activities of HTM in this study,and found that it showed activities against M.luteus,B.thuringiensis and A.baumannii with MIC of 4μg/mL,4μg/mL,and 64μg/mL,respectively.

一个新的忽地笑O-甲基转移酶基因的克隆与原核表达

采用RACE技术克隆得到一个新的忽地笑O-甲基转移酶(OMT)基因,命名为LaOMT。LaOMT的cDNA序列为1 379 bp,开放阅读框1 077 bp,预测编码蛋白包含358个氨基酸。序列比对分析发现,LaOMT编码的蛋白序列具有依赖于S-腺苷-L-甲硫氨酸(SAM)的高度保守的结构域,与葡萄、大豆等其他植物OMTs蛋白的相似性为50%左右。将LaOMT基因连接到原核表达载体pET28a上,转化大肠杆菌Rosetta(DE3)的SDS-PAGE电泳结果显示,重组蛋白受异丙基硫代半乳糖苷(IPTG)诱导表达,分子量大小约为45 kDa,与已报导的其他植物的OMTs蛋白大小基本一致。此外,半定量RT-PCR分析结果表明,LaOMT在忽地笑的叶、鳞茎、根和花中均有表达,其中花中的表达量最低。