酮生物合成路径及其关键酶的研究进展

冯晓晓，刘 越,2 ，孙洪波，张琳霞，李 华，马 徐，唐 丽

(1 中央民族大学 生命与环境科学学院，北京 100081；2 中国中医科学院 中药资源中心，北京 100700)

图1 酮类化合物的基本母核

根据Peres分类法[3]，可将酮类化合物分为5种结构类型：a.简单的氧代酮(含羟基、甲氧基等取代基)，如1-羟基-2,3,5-三甲氧基酮(同时含有羟基和甲氧基取代基)(图2 a-1)，3,4,5,6-四羟基酮(仅含有羟基取代基)(图2 a-2)；b.酮糖苷(含糖基取代基)，根据成苷的原子不同又可分为酮氧苷和酮碳苷2类，如当药醇苷(即1,5-二羟基-3-甲氧基酮-8-O-β-D-吡喃葡萄糖苷，图2 b-1)属于酮氧苷，芒果苷(即2-β-D-吡喃葡萄糖基-1,3,6,7-四羟基酮，图2 b-2)属于酮碳苷；c.异戊烯基酮(含异戊烯基取代基)，如α-倒捻子素(即1,3,6-三羟基-7-甲氧基-2,8-双异戊烯基酮)C2、C8位置上含有2个异戊烯基(图2 c)；d.酮木脂素(由苯丙基和邻位二羟基取代的酮通过二氧杂环己烷连接)[4]，结构式见图2 d，R1～R7指-H，-OH，-OCH33种取代基，不同物质的取代基数量和位置不同，如kielcorins和subalatin 2种物质的结构(图2 d-1，d-2)；e.其他酮类化合物[5]。

图2 酮类化合物的结构类型

图3 酮可能的合成路径Ⅰ

图4 苯甲酸和3-羟基苯甲酸的可能合成路径

Chantarasriwong等[36]阐述了藤黄科植物中酮的可能生物合成路径Ⅱ(图5)，认为高等植物中酮的生物合成起始于莽草酸-乙酸途径(A环来源于乙酸，C环来源于莽草酸途径，图5)，并通过氧化模式获得。由磷酸烯醇式丙酮酸和D-赤藓糖-4-磷酸通过醇醛式缩合形成莽草酸，再经过氧化、脱水、烯醇化形成原儿茶酸，原儿茶酸通过辅酶A的活化形成活化酯，之后与3分子丙二酰辅酶A形成中间产物，烯醇化之后通过分子内的克莱森缩合反应形成桑橙素等二苯甲酮类物质，不同的二苯甲酮类物质来源可能不同，桑橙素通过酚醛耦合可形成1,3,5,6-四羟基酮等酮类化合物。

3.1 3-羟基苯甲酸辅酶A连接酶

3-羟基苯甲酸辅酶A连接酶(3-hydroxybenzoate:CoA ligase，3HBL)是酮生物合成过程中的一种关键酶。酮的生物合成需要3-羟基苯甲酸或苯甲酸为反应物，这2种物质在3-羟基苯甲酸辅酶A连接酶的活化作用下，形成3-羟基苯甲酰辅酶A或苯甲酰辅酶A，才能使酮的生物合成继续进行。在观果金丝桃中，3-羟基苯甲酸辅酶A连接酶的优选底物是3-羟基苯甲酸，同时苯甲酸也是相对有效的催化底物，但是催化活性较低；而在百金花中，3-羟基苯甲酸是其惟一的有效底物[32]。

Barillas等[33]早期研究发现，3-羟基苯甲酸辅酶A连接酶的最适pH为7.0，最适温度为25～30 ℃，酶分子质量大约是50 ku。之后Barillas等[37]通过十二烷基硫酸钠聚丙烯酰胺凝胶电泳，又发现分子质量分别为41.5和40.5 ku的2种多肽，并通过胰蛋白酶消化试验发现这2种蛋白在结构上具有相关性。3-羟基苯甲酸辅酶A连接酶在植物中的研究有限，在细菌上的研究相对较多。NCBI上登录了1条关于固氮弧菌属物种EbN1的3HBL基因(登录号：3179571)和10条细菌的3HBL蛋白序列(登录号：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)。目前未见植物3HBL基因或蛋白序列的报道，相关研究有待进一步深入。

图5 酮可能的合成路径Ⅱ[36]

3.2 二苯甲酮合成酶

二苯甲酮合成酶(Benzophenone synthase，BPS)也是酮生物合成路径中的一种关键酶。研究表明，二苯甲酮是许多高等植物器官中酮生物合成的直接前体物质[38]，在百金花、观果金丝桃等植物中，2,3′,4,6-四羟基二苯甲酮是酮生物合成的直接前体物质[39]。百金花细胞中酮类物质生物合成的关键步骤是C13骨架，如中间体二苯甲酮[40-41]的形成，二苯甲酮合成酶是二苯甲酮形成过程所必需的催化酶，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-羟基苯甲酰辅酶A与3分子丙二酰辅酶A缩合形成2,3′,4,6-四羟基二苯甲酮，或是有效地催化苯甲酰辅酶A与3分子丙二酰辅酶A缩合形成2,4,6-三羟基二苯甲酮，再进一步生成2,3′,4,6-四羟基二苯甲酮[43]。在百金花中，二苯甲酮合成酶以3-羟基苯甲酰辅酶A为底物；在观果金丝桃中，以苯甲酰辅酶A为底物时，二苯甲酮合成酶的活性比以3-羟基苯甲酰辅酶A为底物时高[28]。

二苯甲酮合成酶(BPS)是一种植物类型Ⅲ聚酮合酶(Polyketide synthases，PKS)。植物类型Ⅲ PKS即查尔酮合酶超家族，都是由分子质量为40～45 ku、大小适中的亚基组成的同型二聚体，活性位点为 Cys164、His303 和 Asn336，这3个在植物类型Ⅲ PKS 超家族中绝对保守的氨基酸组成了该类酶的活性中心(三联体活性中心)，即“起始底物分子结合结构域”和“环化反应结构域”[44-45]。植物Ⅲ型PKS基因家族包括查尔酮合成酶(Chalcone synthase，CHS)基因以及由其分化形成的具有新的底物选择性和产物特异性的“类CHS(CHS-Like)基因”[46]。从1983年首次从欧芹中克隆到CHS以来，目前 GenBank上登录的Ⅲ型PKS mRNA全序列已超过1 000条[47]，各种植物类型Ⅲ PKS基因的保守性很强，序列一般由2个外显子和1个内含子组成，个别含有多个内含子。

二苯甲酮合成酶(BPS)基因作为一种“类CHS基因”在个别植物中已被克隆。目前，GenBank上登录的BPS mRNA序列有4条。从金丝桃科植物观果金丝桃(Hypericumandrosaemum)中克隆出了1 398 bp的BPS全长cDNA序列(GenBank登录号：AF352395.1)，其开放阅读框(ORF)长1 188 bp，编码395个氨基酸，形成的酶分子质量大小为42.8 ku，这种酶与从观果金丝桃中克隆出的查尔酮合成酶的一致性为60.1%，与查尔酮合酶超家族其他成员的一致性为53%～63%[48]。He等[49]通过金丝桃科贯叶金丝桃(Hypericumperforatum)转录组数据的分析，比对注释得到BPS基因。目前，从贯叶金丝桃中已克隆出长1 185 bp的BPS部分mRNA序列(GenBank登录号：EF507429.1)。从金丝桃科植物元宝草(Hypericumsampsonii)中已克隆出二苯甲酮合成酶(HsBPS)cDNA(GenBank登录号：JQ670939.1)，其开放阅读框长1 188 bp，编码395个氨基酸，形成分子质量大小为42.7 ku的酶，此酶与从元宝草中克隆出的查尔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]，且在元宝草根中表达量丰富。从藤黄科植物莽吉柿(Garciniamangostana)中已克隆出二苯甲酮合成酶(GmBPS)(GenBank登录号：JF907623.1)基因，其开放阅读框长1 176 bp，编码391个氨基酸，形成分子质量大小为42.7 ku的酶，此酶与观果金丝桃BPS和元宝草BPS分别表现出78%和77%的同源性[30]。目前，还未见龙胆科植物中有关BPS克隆的报道。

细胞色素氧化酶P450酶系(Cytochrome P450s，CYPs)是一组结构和功能相关的超家族基因编码的同工酶，主要存在于生物体的内质网内，是混合功能氧化酶中最重要的一种酶系[51]。植物CYPs的功能主要可归为2大类：参与生物合成途径和生物解毒途径。研究发现，龙胆科植物川西獐牙菜中烯醚萜类化合物生物合成的第1个限速酶是香叶醇10-羟化酶，这种酶是一种细胞色素P450单氧酶CYP76B10，属于CYP76亚家族[52]。而酮合成酶的分属家族目前还未明确，也未见关于酮合成酶基因的研究，需要进一步探索。

4 前景和展望

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