酮生物合成路徑及其關(guān)鍵酶的研究進(jìn)展

馮曉曉，劉 越,2 ，孫洪波，張琳霞，李 華，馬 徐，唐 麗

(1 中央民族大學(xué) 生命與環(huán)境科學(xué)學(xué)院，北京 100081；2 中國中醫(yī)科學(xué)院 中藥資源中心，北京 100700)

圖1 酮類化合物的基本母核

根據(jù)Peres分類法[3]，可將酮類化合物分為5種結(jié)構(gòu)類型：a.簡單的氧代酮(含羥基、甲氧基等取代基)，如1-羥基-2,3,5-三甲氧基酮(同時(shí)含有羥基和甲氧基取代基)(圖2 a-1)，3,4,5,6-四羥基酮(僅含有羥基取代基)(圖2 a-2)；b.酮糖苷(含糖基取代基)，根據(jù)成苷的原子不同又可分為酮氧苷和酮碳苷2類，如當(dāng)藥醇苷(即1,5-二羥基-3-甲氧基酮-8-O-β-D-吡喃葡萄糖苷，圖2 b-1)屬于酮氧苷，芒果苷(即2-β-D-吡喃葡萄糖基-1,3,6,7-四羥基酮，圖2 b-2)屬于酮碳苷；c.異戊烯基酮(含異戊烯基取代基)，如α-倒捻子素(即1,3,6-三羥基-7-甲氧基-2,8-雙異戊烯基酮)C2、C8位置上含有2個(gè)異戊烯基(圖2 c)；d.酮木脂素(由苯丙基和鄰位二羥基取代的酮通過二氧雜環(huán)己烷連接)[4]，結(jié)構(gòu)式見圖2 d，R1～R7指-H，-OH，-OCH33種取代基，不同物質(zhì)的取代基數(shù)量和位置不同，如kielcorins和subalatin 2種物質(zhì)的結(jié)構(gòu)(圖2 d-1，d-2)；e.其他酮類化合物[5]。

圖2 酮類化合物的結(jié)構(gòu)類型

圖3 酮可能的合成路徑Ⅰ

圖4 苯甲酸和3-羥基苯甲酸的可能合成路徑

Chantarasriwong等[36]闡述了藤黃科植物中酮的可能生物合成路徑Ⅱ(圖5)，認(rèn)為高等植物中酮的生物合成起始于莽草酸-乙酸途徑(A環(huán)來源于乙酸，C環(huán)來源于莽草酸途徑，圖5)，并通過氧化模式獲得。由磷酸烯醇式丙酮酸和D-赤蘚糖-4-磷酸通過醇醛式縮合形成莽草酸，再經(jīng)過氧化、脫水、烯醇化形成原兒茶酸，原兒茶酸通過輔酶A的活化形成活化酯，之后與3分子丙二酰輔酶A形成中間產(chǎn)物，烯醇化之后通過分子內(nèi)的克萊森縮合反應(yīng)形成桑橙素等二苯甲酮類物質(zhì)，不同的二苯甲酮類物質(zhì)來源可能不同，桑橙素通過酚醛耦合可形成1,3,5,6-四羥基酮等酮類化合物。

3.1 3-羥基苯甲酸輔酶A連接酶

3-羥基苯甲酸輔酶A連接酶(3-hydroxybenzoate:CoA ligase，3HBL)是酮生物合成過程中的一種關(guān)鍵酶。酮的生物合成需要3-羥基苯甲酸或苯甲酸為反應(yīng)物，這2種物質(zhì)在3-羥基苯甲酸輔酶A連接酶的活化作用下，形成3-羥基苯甲酰輔酶A或苯甲酰輔酶A，才能使酮的生物合成繼續(xù)進(jìn)行。在觀果金絲桃中，3-羥基苯甲酸輔酶A連接酶的優(yōu)選底物是3-羥基苯甲酸，同時(shí)苯甲酸也是相對(duì)有效的催化底物，但是催化活性較低；而在百金花中，3-羥基苯甲酸是其惟一的有效底物[32]。

Barillas等[33]早期研究發(fā)現(xiàn)，3-羥基苯甲酸輔酶A連接酶的最適pH為7.0，最適溫度為25～30 ℃，酶分子質(zhì)量大約是50 ku。之后Barillas等[37]通過十二烷基硫酸鈉聚丙烯酰胺凝膠電泳，又發(fā)現(xiàn)分子質(zhì)量分別為41.5和40.5 ku的2種多肽，并通過胰蛋白酶消化試驗(yàn)發(fā)現(xiàn)這2種蛋白在結(jié)構(gòu)上具有相關(guān)性。3-羥基苯甲酸輔酶A連接酶在植物中的研究有限，在細(xì)菌上的研究相對(duì)較多。NCBI上登錄了1條關(guān)于固氮弧菌屬物種EbN1的3HBL基因(登錄號(hào)：3179571)和10條細(xì)菌的3HBL蛋白序列(登錄號(hào)：WP_004358116.1，ENO98131.1，CAC28158.1，YP_157393.1，WP_018988708.1，WP_006116890.1，WP_011236227.1，EGE47156.1，CAI06492.1，Q9AJS8.1)。目前未見植物3HBL基因或蛋白序列的報(bào)道，相關(guān)研究有待進(jìn)一步深入。

圖5 酮可能的合成路徑Ⅱ[36]

3.2 二苯甲酮合成酶

二苯甲酮合成酶(Benzophenone synthase，BPS)也是酮生物合成路徑中的一種關(guān)鍵酶。研究表明，二苯甲酮是許多高等植物器官中酮生物合成的直接前體物質(zhì)[38]，在百金花、觀果金絲桃等植物中，2,3′,4,6-四羥基二苯甲酮是酮生物合成的直接前體物質(zhì)[39]。百金花細(xì)胞中酮類物質(zhì)生物合成的關(guān)鍵步驟是C13骨架，如中間體二苯甲酮[40-41]的形成，二苯甲酮合成酶是二苯甲酮形成過程所必需的催化酶，它能催化二苯甲酮和酮C13骨架的形成[42]。此酶能有效地催化3-羥基苯甲酰輔酶A與3分子丙二酰輔酶A縮合形成2,3′,4,6-四羥基二苯甲酮，或是有效地催化苯甲酰輔酶A與3分子丙二酰輔酶A縮合形成2,4,6-三羥基二苯甲酮，再進(jìn)一步生成2,3′,4,6-四羥基二苯甲酮[43]。在百金花中，二苯甲酮合成酶以3-羥基苯甲酰輔酶A為底物；在觀果金絲桃中，以苯甲酰輔酶A為底物時(shí)，二苯甲酮合成酶的活性比以3-羥基苯甲酰輔酶A為底物時(shí)高[28]。

二苯甲酮合成酶(BPS)是一種植物類型Ⅲ聚酮合酶(Polyketide synthases，PKS)。植物類型Ⅲ PKS即查爾酮合酶超家族，都是由分子質(zhì)量為40～45 ku、大小適中的亞基組成的同型二聚體，活性位點(diǎn)為 Cys164、His303 和 Asn336，這3個(gè)在植物類型Ⅲ PKS 超家族中絕對(duì)保守的氨基酸組成了該類酶的活性中心(三聯(lián)體活性中心)，即“起始底物分子結(jié)合結(jié)構(gòu)域”和“環(huán)化反應(yīng)結(jié)構(gòu)域”[44-45]。植物Ⅲ型PKS基因家族包括查爾酮合成酶(Chalcone synthase，CHS)基因以及由其分化形成的具有新的底物選擇性和產(chǎn)物特異性的“類CHS(CHS-Like)基因”[46]。從1983年首次從歐芹中克隆到CHS以來，目前 GenBank上登錄的Ⅲ型PKS mRNA全序列已超過1 000條[47]，各種植物類型Ⅲ PKS基因的保守性很強(qiáng)，序列一般由2個(gè)外顯子和1個(gè)內(nèi)含子組成，個(gè)別含有多個(gè)內(nèi)含子。

二苯甲酮合成酶(BPS)基因作為一種“類CHS基因”在個(gè)別植物中已被克隆。目前，GenBank上登錄的BPS mRNA序列有4條。從金絲桃科植物觀果金絲桃(Hypericumandrosaemum)中克隆出了1 398 bp的BPS全長cDNA序列(GenBank登錄號(hào)：AF352395.1)，其開放閱讀框(ORF)長1 188 bp，編碼395個(gè)氨基酸，形成的酶分子質(zhì)量大小為42.8 ku，這種酶與從觀果金絲桃中克隆出的查爾酮合成酶的一致性為60.1%，與查爾酮合酶超家族其他成員的一致性為53%～63%[48]。He等[49]通過金絲桃科貫葉金絲桃(Hypericumperforatum)轉(zhuǎn)錄組數(shù)據(jù)的分析，比對(duì)注釋得到BPS基因。目前，從貫葉金絲桃中已克隆出長1 185 bp的BPS部分mRNA序列(GenBank登錄號(hào)：EF507429.1)。從金絲桃科植物元寶草(Hypericumsampsonii)中已克隆出二苯甲酮合成酶(HsBPS)cDNA(GenBank登錄號(hào)：JQ670939.1)，其開放閱讀框長1 188 bp，編碼395個(gè)氨基酸，形成分子質(zhì)量大小為42.7 ku的酶，此酶與從元寶草中克隆出的查爾酮合成酶序列在基因和氨基酸水平上均有57.0%的一致性[50]，且在元寶草根中表達(dá)量豐富。從藤黃科植物莽吉柿(Garciniamangostana)中已克隆出二苯甲酮合成酶(GmBPS)(GenBank登錄號(hào)：JF907623.1)基因，其開放閱讀框長1 176 bp，編碼391個(gè)氨基酸，形成分子質(zhì)量大小為42.7 ku的酶，此酶與觀果金絲桃BPS和元寶草BPS分別表現(xiàn)出78%和77%的同源性[30]。目前，還未見龍膽科植物中有關(guān)BPS克隆的報(bào)道。

細(xì)胞色素氧化酶P450酶系(Cytochrome P450s，CYPs)是一組結(jié)構(gòu)和功能相關(guān)的超家族基因編碼的同工酶，主要存在于生物體的內(nèi)質(zhì)網(wǎng)內(nèi)，是混合功能氧化酶中最重要的一種酶系[51]。植物CYPs的功能主要可歸為2大類：參與生物合成途徑和生物解毒途徑。研究發(fā)現(xiàn)，龍膽科植物川西獐牙菜中烯醚萜類化合物生物合成的第1個(gè)限速酶是香葉醇10-羥化酶，這種酶是一種細(xì)胞色素P450單氧酶CYP76B10，屬于CYP76亞家族[52]。而酮合成酶的分屬家族目前還未明確，也未見關(guān)于酮合成酶基因的研究，需要進(jìn)一步探索。

4 前景和展望

[參考文獻(xiàn)]

[1] 吳立軍.天然產(chǎn)物化學(xué) [M].4版.北京:人民衛(wèi)生出版社,2003:175.

Wu L J.Natural product chemistry [M].4th ed.Beijing:People’s Medical Publishing House,2003:175.(in Chinese)

Ma C.Distribution and pharmacological function of xanthones in plants [J].Journal of Anhui Agricultural Sciences,2009,37(31):15244-15245.(in Chinese)

[3] Peres V,Nagem T J,Oliveira F F.Tetraoxygenated naturally occurring xanthones [J].Phytochemistry,2000,55(7):683-710.

[4] 毋 艷.紅厚殼、薄葉紅厚殼和泰山赤靈芝菌絲體化學(xué)成分及生物活性研究 [D].北京:中國協(xié)和醫(yī)科大學(xué),2004.

Wu Y.Chemical constituents and bioactivity ofCalophylluminophyllum,Calophyllummembranaceumand mycelium ofGanodermalucidum[D].Beijing:Chinese Peking Union Medical College,2004.(in Chinese)

Wang Y,Wang S S,Zhou D H,et al.Spectral characteristics of phytoxanthones [J].Natural Product Research and Development,2002,14(5):85-89.(in Chinese)

Xing C,Xu B,Guo W,et al.Protection of 1-hydroxy-2,3,5-trimethoxyxanthone on acute lung injury of mice induced by lipopolysaccharide [J].Chinese Traditional and Herbal Drugs,2007,37(9):1355-1359.(in Chinese)

Dai Z,Hu C P,Wu T,et al.Effects of 3,4,5,6-tetrahydroxyxanthone on myocardial ischemia-reperfusion injury in isolated rat hearts [J].Chinese Journal of Clinical Pharmacology and Therapeutics,2005,10(5):532-535.(in Chinese)

[8] 王煥弟,譚玉成,白雪芳,等.藏藥濕生扁蕾的抑菌作用研究 [J].時(shí)珍國醫(yī)國藥,2006,17(10):1901-1902.

Wang H D,Tan Y C,Bai X F,et al.Studies of the antimicrobial activity of gentianopsis paludosa [J].Lishizhen Medicine and Materia Medica Research,2006,17(10):1901-1902.(in Chinese)

[9] Li J F,Lu G F,Zou Y Y.Demethylbellidifolin inhibits proliferation and activation of hepatic stellate cells [J].Journal of Investigative Surgery,2011,24(4):171-177.

[10] Rivera D G,Balmaseda I H,León A A,et al.Anti-allergic properties ofMangiferaindicaL.extract (Vimang) and contribution of its glucosylxanthone mangiferin [J].Journal of Pharmacy and Pharmacology,2006,58(3):385-392.

[11] Rao V S,Carvalho A C,Trevisan M T,et al.Mangiferin ameliorates 6-hydroxydopamine-induced cytotoxicity and oxidative stress in ketamine model of schizophrenia [J].Pharmacological Reports,2012,64(4):848-856.

[12] Azevedo C M,Afonso C M,Sousa D,et al.Multidimensional optimization of promising antitumor xanthone derivatives [J].Bioorganic & Medicinal Chemistry,2013,21(11):2941-2959.

[13] Teh S S,Ee G C,Mah S H,et al.Cytotoxicity and structure-activity relationships of xanthone derivatives fromMesuabeccariana,MesuaferreaandMesuacongestifloratowards nine human cancer cell lines [J].Molecules,2013,18(2):1985-1994.

[14] Mahabusarakam W,Proudfoot J,Taylor W,et al.Inhibition of lipoprotein oxidation by prenylated xanthones derived from mangostin [J].Free Radical Research,2000,33(5):643-659.

[15] Szkaradek N,Rapacz A,Pytka K,et al.Synthesis and preliminary evaluation of pharmacological properties of some piperazine derivatives of xanthone [J].Bioorganic & Medicinal Chemistry,2013,21(2):514-522.

[16] Winter D K,Sloman D L,Porco J A J.Polycyclic xanthone natural products:Structure,biological activity and chemical synthesis [J].Natural Product Reports,2013,30(3):382-391.

[17] Hu H,Liao H,Zhang J,et al.First identification of xanthone sulfonamides as potent acyl-CoA:Cholesterol acyltransferase (ACAT) inhibitors [J].Bioorganic & Medicinal Chemistry Letters,2010,20(10):3094-3097.

[18] 唐 麗,金振南,門美佳,等.藏藥藏茵陳的研究進(jìn)展及開發(fā)利用 [J].中央民族大學(xué)學(xué)報(bào):自然科學(xué)版,2007,16(2):176-178.

Tang L,Jin Z N,Men M J,et al.Recent progress and utilization of Tibetan medicine zangyinchen [J].Journal of the Central University for Nationalities:Natural Sciences Edition,2007,16(2):176-178.(in Chinese)

[19] Du X G,Wang W,Zhang Q Y,et al.Identification of xanthones fromSwertiapuniceausing high-performance liquid chromatography coupled with electrospray ionization tandem mass spectrometry [J].Rapid Communications in Mass Spectrometry,2012,26(24):2913-2923.

[20] 王洪玲,耿長安,張雪梅,等.大籽獐牙菜化學(xué)成分的研究 [J].中國中藥雜志,2010,35(23):3161-3164.

Wang H L,Geng C A,Zhang X M,et al.Chemical constituents ofSwertiamacrosperma[J].China Journal of Chinese Materia Medica,2010,35(23):3161-3164.(in Chinese)

[21] Suryawanshi S,Mehrotra N,Asthana R K,et al.Liquid chromatography/tandem mass spectrometric study and analysis of xanthone and secoiridoid glycoside composition ofSwertiachirata,a potent antidiabetic [J].Rapid Communications in Mass Spectrometry,2006,20(24):3761-3768.

[22] 孫洪發(fā),胡伯林,丁經(jīng)業(yè),等.川西獐牙菜甙類成分 [J].植物學(xué)報(bào),1991,33(1):31-37.

Sun H F,Hu B L,Ding J Y,et al.The glucosides fromSwertiamussotiiFranch [J].Journal of Integrative Plant Biology,1991,33(1):31-37.(in Chinese)

Xu X D,Yang J S.Studies on chemical constituents ofSwertiapubescens[J].Chinese Pharmaceutical Journal,2005,40(9):657-659.(in Chinese)

[24] Liu X,Liu Y,Chen J,et al.Simultaneous analysis of xanthone glycosides inHaleniaellipticaby HPLC-DAD-ESI-MS [J].Journal of Chromatographic Science,2010,48(1):76-80.

[25] 羅洲飛,劉妮娜,徐彥軍,等.扁蕾不同部位的化學(xué)成分研究 [J].湖南農(nóng)業(yè)科學(xué),2012(9):99-102.

Luo Z F,Liu N N,Xu Y J,et al.Chemical constituents of different parts ofGentianopsisbarbata(Froel.) Ma [J].Hunan Agricultural Sciences,2012(9):99-102.(in Chinese)

[26] Qiao Y,Yuan Y,Cui B,et al.New xanthone glycosides fromComastomapedunculatum[J].Natural Product Chemistry,2012,78(14):1591-1596.

[27] Sanchez J F,Entwistle R,Hung J H,et al.Genome-based deletion analysis reveals the prenyl xanthone biosynthesis pathway inAspergillusnidulans[J].Journal of the American Chemical Society,2011,133(11):4010-4017.

[28] Schmidt W,Beerhues L.Alternative pathways of xanthone biosynthesis in cell cultures ofHypericumandrosaemumL. [J].FEBS Letters,1997,420(2/3):143-146.

[29] Schmidt W,Peters S,Beerhues L.Xanthone 6-hydroxylase fr-om cell cultures ofCentauriumerythraeaRAFN andHypericumandrosaemumL. [J].Phytochemistry,2000,53(4):427-431.

[30] Nualkaew N,Morita H,Shimokawa Y,et al.Benzophenone sy-nthase fromGarciniamangostanaL.pericarps [J].Phytochemistry,2012,77:60-69.

[31] Gaid M M,Sircar D,Müller A,et al.Cinnamate:CoA ligase initiates the biosynthesis of a benzoate-derived xanthone phytoalexin inHypericumcalycinumcell cultures [J].Plant Physiology,2012,160(3):1267-1280.

[32] Peters S,Schmidt W,Beerhues L.Regioselective oxidative ph-enol couplings of 2,3′,4,6-tetrahydroxybenzophenone in cell cultures ofCentauriumerythraeaRAFN andHypericumandrosaemumL. [J].Planta,1997,204(1):64-69.

[33] Barillas W,Beerhues L.3-Hydroxybenzoate:Coenzyme A ligase and 4-coumarate:Coenzyme A ligase from cultured cells ofCentauriumerythraea[J].Planta,1997,202(1):112-116.

[34] Wang C Z,Maier U H,Keil M,et al.Phenylalanine-independent biosynthesis of 1,3,5,8-tetrahydroxyxanthone [J].European Journal of Biochemistry,2003,270(14):2950-2958.

[35] Abd El-Mawla A M A,Beerhues L.Benzonic acid biosynthesis in cell cultures ofHypericumandrosaemum[J].Planta,2002,214(5):727-733.

[36] Chantarasriwong O,Batova A,Chavasiri W,et al.Chemistry and biology of the caged garcinia xanthones [J].Chemistry,2010,16(33):9944-9962.

[37] Barillas W,Beerhues L.3-Hydroxybenzoate:Coenzyme A ligase from cell cultures ofCentauriumerythraea:Isolation and characterization [J].Biological Chemistry,2000,381(2):155-160.

[38] Fujita M,Inoue T.Further studies on the biosynthesis of ma-ngiferin inAnemarrhenaasphodeloides:Hydroxylation of the shikimate-derived ring [J].Phytochemistry,1981,20(9):2183-2185.

[39] Bennett G J,Lee H H.Xanthones from guttiferae [J].Phytochemistry,1989,28(4):967-998.

[40] Beerhues L,Berger U.Xanthones in cell suspension cultures of twoCentauriumspecies[J].Phytochemistry,1994,35(5):1227-1231.

[41] Beerhues L,Berger U.Differential accumulation of xanthones in methyl-jasmonate and yeast-extract-treated cell cultures ofCentauriumerythraeaandCentauriumlittorale[J].Planta,1995,197(4):608-612.

[42] Beerhues L,Liu B.Biosynthesis of biphenyls and benzophenones:Evolution of benzoic acid-specific type Ⅲ polyketide synthases in plants [J].Phytochemistry,2009,70(15/16):1719-1727.

[43] Beerhues L.Benzophenone synthase from cultured cells ofCentauriumerythraea[J].FEBS Letters,1996,383(3):264-266.

[44] 馬蘭青,師光祿,葉和春,等.植物類型Ⅲ聚酮合酶超家族基因結(jié)構(gòu)、功能及代謝產(chǎn)物 [J].生物工程學(xué)報(bào),2010,26(11):1482-1492.

Ma L Q,Shi G L,Ye H C,et al.Plant-specific type Ⅲ polyketide synthase superfamily:Gene structure,function and metabolistes [J].Chinese Journal of Biotechnology,2010,26(11):1482-1492.(in Chinese)

[45] Weng J K,Noel J P.Structure-function analyses of plant type Ⅲ polyketide synthases [J].Methods in Enzymology,2012,515:317-335.

[46] 楊 繼,顧紅雅.查爾酮合酶超家族(chalcone synthase superfamily)基因重復(fù)和分化樣式 [J].科學(xué)通報(bào),2006,51(7):745-749.

Yang J,Gu H Y.Gene duplication and differentiation of chalcone synthase superfamily [J].Chinese Science Bulletin,2006,51(7):745-749.(in Chinese)

[47] 生書晶,趙樹進(jìn).植物Ⅲ型聚酮合酶的分子機(jī)制與應(yīng)用前景 [J].生物工程學(xué)報(bào),2009,25(11):1601-1607.

Sheng S J,Zhao S J.A comprehensive overview of type Ⅲ polyketide synthases from plants:Molecular mechanism and application perspective [J].Chinese Journal of Biotechnology,2009,25(11):1601-1607.(in Chinese)

[48] Liu B,Falkenstein-Paul H,Schmidt W,et al.Benzophenone synthase and chalcone synthase fromHypericumandrosaemumcell cultures:cDNA cloning,functional expression,and site-directed mutagenesis of two polyketide synthases [J].The Plant Journal,2003,34(6):847-855.

[49] He M,Wang Y,Hua W P,et al.De novo sequencing ofHypericumperforatumtranscriptome to identify potential genes involved in the biosynthesis of active metabolites [J].Plos One,2012,7(7):e42081(1-10).

[50] Huang L,Wang H,Ye H,et al.Differential expression of benzophenone synthase and chalcone synthase inHypericumsampsonii[J].Natural Product Communications,2012,7(12):1615-1618.

[51] 劉移民,王翔樸.細(xì)胞色素氧化酶P450研究新進(jìn)展 [J].衛(wèi)生毒理學(xué)雜志,2000,14(4):243-246.

Liu Y M,Wang X P.New research progress of cytochrome oxidase P450 [J].Journal of Health Toxicology,2000,14(4):243-246.(in Chinese)

[52] Wang J,Liu Y,Cai Y,et al.Cloning and functional analysis of geraniol 10-hydroxylase,a cytochrome P450 fromSwertiamussotiiFranch [J].Bioscience,Biotechnology,and Biochemistry,2010,74(8):1583-1590.