崔一芳^,1, 郑敏², 丁双阳², 朱奎^,21北京市农林科学院畜牧兽医研究所/畜禽疫病防控技术北京市重点实验室,北京 100097
²中国农业大学动物医学院/国家兽药安全评价中心,北京 100193

Advances of Biosynthesis and Toxicity of Cereulide Produced by Emetic Bacillus cereus

CUI YiFang^,1, ZHENG Min², DING ShuangYang², ZHU Kui^,21Beijing Key Laboratory for Prevention and Control of Infectious Diseases in Livestock and Poultry/Institute of Animal Husbandry and Veterinary Medicine, Beijing Academy of Agricultural and Forestry Sciences, Beijing 100097
²National Center for Veterinary Drug Evaluation/College of Veterinary Medicine, China Agricultural University, Beijing 100193

通讯作者: 朱奎,Tel：010-62733695;E-mail：zhuk@cau.edu.cn

责任编辑: 林鉴非
收稿日期:2020-05-6接受日期:2020-09-27网络出版日期:2021-06-16

基金资助:

国家重点研发计划.2017YFC1600305 奶牛产业技术体系北京市创新团队 北京市农林科学院创新能力建设项目.KJCX20161503

Received:2020-05-6Accepted:2020-09-27Online:2021-06-16
作者简介 About authors
崔一芳,Tel：010-51503195;E-mail：cuiyf_baafs@163.com。






摘要
蜡样芽孢杆菌是一类兼性厌氧的革兰氏阳性杆菌,可以产生芽孢抵抗不良环境,并广泛存在于土壤、水、空气和多种食物中。致病性蜡样芽孢杆菌是常见的食源性条件致病菌之一,其引发的食物中毒主要是由其产生的毒素导致的。致吐毒素cereulide是致病性蜡样芽孢杆菌产生的重要毒素之一,是一种小分子亲脂性十二环肽,结构性质十分稳定。Cereulide能够引起恶心、呕吐等轻微的食物中毒症状,也可导致如肝性脑病、急性肝脏衰竭等严重致死的疾病。当前对于cereulide的毒性作用机制研究局限于其刺激传入迷走神经引起呕吐症状,以及作为钾离子载体,诱导线粒体膜电位丧失,并最终导致细胞死亡,而对于其导致的严重肝脏和脑部病变的毒性作用机制研究仍十分不足。Cereulide是由cereulide合成酶基因簇(ces)编码,非核糖体肽合成酶(nonribosomal peptide-synthetase, NRPS)系统控制合成的。Cereulide由两个羟基酸和两个氨基酸残基[-D-HIC-D-Ala-L-HIV-L-Val-]经3次迭代组成三聚体缩酚酞,其结构特殊并有很强的代表性,但因NRPS合成系统的灵活性会产生许多变体,因此cereulide的毒性与其生物合成过程息息相关。文章在现有文献报道和研究数据的基础上,总结并提出了cereulide的生物合成机理：首先,cereulide合成基因簇的CesA和CesB结构域分别识别D-α-酮羧酸、L-丙氨酸、L-α-酮异戊酸和L-缬氨酸,通过共价结合形成cereulide的主要合成单元二肽;其次,依照上述过程重复合成四肽;再通过重复反应合成第二个四肽,两个四肽通过酯化形成八肽;再次重复上述反应,形成三元络合的产物肽;最后,由于ces-NRPS的硫酯酶结构域活性中心表面结构阻止外部水分子进入,并诱导内部亲核攻击反应,最终释放出环状cereulide。目前,由产cereulide的蜡样芽孢杆菌引起的食物中毒风险被低估。并且,本团队前期研究发现部分芽孢杆菌微生态制剂中混有产cereulide的蜡样芽孢杆菌菌株。因此,产cereulide蜡样芽孢杆菌的存在对食品安全和公共健康均构成了潜在风险。文章综述了cereulide的毒性作用及机制,为进一步研发cereulide防控措施提供科学依据;总结并提出了cereulide的生物合成机理,强调了催化酮酸形成酯的酮还原酶域(KR),及形成重复单元和环肽的硫酯酶域(TE)在其合成中的重要作用,为阐明类似结构的非核糖体肽合成提供新的思路。
关键词： 蜡样芽孢杆菌;致吐毒素cereulide;生物合成;非核糖体肽合成酶;毒性

Abstract
Bacillus cereus (B. cereus) is a gram-positive facultative anaerobe that can produce spores to survive adverse environments. And it is widely present in soil, water, air and a variety of foods. Pathogenic B. cereus is one of the most common food-borne pathogens, and the toxins produced by B. cereus are the main cause of food poisoning. Cereulide is a major toxin produced by pathogenic B. cereus, which is a small molecule lipophilic cyclic dodecadepsipeptide with stable structural properties. Cereulide can cause mild food poisoning with emetic symptoms, such as nausea and vomiting, and it may induce severe fatal diseases such as hepatic encephalopathy or acute liver failure. Current researches believed that cereulide caused vomiting by stimulating the vagus nerve, and induced the loss of mitochondrial membrane potential by acting as a potassium ionophore, which ultimately led to cell death. However, the toxic mechanism of hepatic encephalopathy or acute liver failure caused by cereulide remains unclear. Cereulide is encoded by the cereulide synthetase gene cluster (ces) and is synthesized by the non-ribosomal peptide synthetase (NRPS) system. Cereulide is composed by two hydroxy acids and two amino acid residues [-D-HIC-D-Ala-L-HIV-L-Val-], which forms a trimer phenolphthalein after three iterations and shows a structural specificity and representativeness. However, isocereulides may be produced due to the flexibility of the NRPS system. Therefore, the toxicity of cereulide is closely related to its biosynthesis process. Based on previous studies, this review summarized and proposed the biosynthesis mechanism of cereulide. Firstly, the CesA and CesB domained in ces recognize D-α-ketocarboxylic acid, L-alanine, L-α-ketoisovalerate and L-valine, respectively, which formed the main synthetic unit dipeptide of cereulide by covalent bonding. Secondly, a tetrapeptide was synthesized by repeating the above process. Thirdly, the second tetrapeptide was synthesized through repeated reactions, and the two tetrapeptides formed an octapeptide through esterification. Fourthly, the above reaction was repeated to form a ternary complex product peptide. Lastly, because the surface structure of the active center of the thioesterase domain in ces-NRPS prevented external water molecules from entering, it induced an internal nucleophilic attack reaction and finally released a circular cereulide. The risk of food poisoning caused by cereulide producing B. cereus was underestimated. In addition, our previous studies have found that some probiotic Bacillus products were contaminated with cereulide-producing B. cereus strains. This posed a potential risk to food safety and public health. This review briefly summarized the characteristics and toxic mechanisms of cereulide, which would provide a scientific basis for the prevention of cereulide. This review also summarized and proposed the biosynthesis process of cereulide. Functions of two domains in the synthesis process need to be focused. The main function of ketoreductase (KR) domain was that it could catalyze the formation of esters of keto acid at the beginning of the biosynthesis processes. The important role of thioesterase (TE) domain was to form repeating units and the cyclic peptide in the last link of synthesis. These could serve as a model for other cyclic peptides synthesized by the non-ribosomal peptide synthetase system.
Keywords：Bacillus cereus;cereulide;biosynthesis;non-ribosomal peptide synthetase;toxicity


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本文引用格式
崔一芳, 郑敏, 丁双阳, 朱奎. 蜡样芽孢杆菌致吐毒素的毒性作用与生物合成研究进展[J]. 中国农业科学, 2021, 54(12): 2666-2674 doi:10.3864/j.issn.0578-1752.2021.12.016
CUI YiFang, ZHENG Min, DING ShuangYang, ZHU Kui. Advances of Biosynthesis and Toxicity of Cereulide Produced by Emetic Bacillus cereus[J]. Scientia Acricultura Sinica, 2021, 54(12): 2666-2674 doi:10.3864/j.issn.0578-1752.2021.12.016


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蜡样芽孢杆菌是一种在自然环境中广泛分布,可产生芽孢的革兰氏阳性杆菌。致病性蜡样芽孢杆菌可产生多种毒素,是引起食物中毒的常见食源性条件致病菌之一^[1]。致吐毒素cereulide是蜡样芽孢杆菌分泌的主要毒素之一,能够引发以恶心和呕吐为主要临床症状的食物中毒,严重的可导致急性肝脏衰竭、肝性脑病,并致死^[2,3,4]。由cereulide引起的食物中毒症状因与金黄色葡萄球菌肠毒素导致的临床症状相似,且症状轻微、病程较短,因而被低估和忽视^[5]。实际上,美国报告了1998—2008年,由蜡样芽孢杆菌引发了235起食源性疾病^[6]。法国统计了2007—2014年发生的140起食源性疾病,其中74起与蜡样芽孢杆菌相关,是仅次于金黄色葡萄球菌的第二大常见食源性病原菌,并且产生cereulide的蜡样芽孢杆菌检出率为16%^[7]。同样德国2007—2013年食源性疾病的调查结果显示,产cereulide的蜡样芽孢杆菌引起的食物中毒风险被低估^[8]。目前对于cereulide的毒性作用机制研究局限于其刺激传入迷走神经引起呕吐症状,以及作为钾离子载体,诱导线粒体膜电位丧失,并最终导致细胞死亡^[9],而对于其导致的严重肝脏和脑部病变的毒性作用机制研究仍十分不足。

另一方面,cereulide是由非核糖体肽合成酶（nonribosomal peptide-synthetase, NRPS）系统控制合成的一种亲脂性小分子十二环肽,结构性质十分稳定,并受基因及多种调节因子控制^[1]。当前对于cereulide导致的食物中毒症状并没有良好的预防和治疗方法^[10,11],一般采用添加防腐剂抑制蜡样芽孢杆菌生长的方法保障食品安全,但cereulide可在菌株被抑制前合成,并且一旦产生就难以被消除^[12],因此缺乏针对cereulide的有效防控策略。此外,目前对于cereulide的生物合成过程,如合成基因簇各结构域功能和调节因子的具体作用均不够明确。

值得注意的是,部分蜡样芽孢杆菌作为益生菌被广泛应用于人类医疗保健、畜牧业、农业和水产环境等多个领域^[13]。例如,美国食品药品监督管理局（Food and Drug Administration, FDA）和饲料工业协会（American Feed Industry Association, AFIA）在1989年就批准了蜡样芽孢杆菌可作为直接应用于畜牧生产的菌种之一^[14]。我国农业农村部1996年也正式批准蜡样芽孢杆菌可作为饲料级微生物添加剂的菌种之一^[15]。尽管目前益生菌的总体使用记录显示出较令人满意的安全性结果,但相对于其应用的广泛度来说,益生菌的安全性并未受到充分鉴定^[16]。例如,本团队前期对益生芽孢杆菌的毒性和耐药性进行了系统评估,发现菌株可产生多种毒素,其中便包括cereulide^[17,18]。此外,详细综述了蜡样芽孢杆菌可能产生的多种毒素,包括毒素特征、毒性作用、合成机制及检测方法,评估其对蜡样芽孢杆菌作为益生菌使用的潜在危害^[19]。

本综述旨在针对蜡样芽孢杆菌产生的主要毒素cereulide,整理其毒性作用机制研究进展,为进一步研发cereulide防控措施提供科学依据;总结并提出其生物合成机理,对其关键结构域在合成过程中的功能进行了补充,为阐明类似结构的非核糖体肽合成提供新的思路。

1 Cereulide的毒性特征

Cereulide是一种由两个羟基酸和两个氨基酸残基[-D-HIC-D-Ala-L-HIV-L-Val-]经3次重复组成的三聚体环状缩酚酞,分子量约为1 152 Da^[20,21]。cereulide的环状结构外部为脂溶性,能够与细胞膜结合;内部为亲水性,中间存在容纳钾离子的腔隙,可进行钾离子的跨膜转运^[9]。cereulide是目前已知最强的钾离子螯合剂,对动物和人体细胞具有普遍的毒性作用^[22]。此外,cereulide性质十分稳定,能够耐高温、耐酸碱,对各种蛋白酶水解作用都有一定的抵抗力,经过食品加工或者胃肠道消化仍可保持活性^[23,24]。例如,cereulide在121℃处理2 h后仍可保持生物活性,并对pH 2.0至pH 11.0的环境耐受^[25]。研究表明,灭活cereulide不仅需要高温处理,还依赖于pH条件,如在pH 9.5的条件下,灭活6 μg·mL^-1 cereulide需在121℃暴露80min或在150℃暴露60 min^[26]。

目前统计发现,环境中仅有1%—2%的蜡样芽孢杆菌菌株会产生cereulide^[27],但在食品和临床样本中产cereulide菌株的流行率却高达32.8%^[8]。产cereulide的菌株通常存在于淀粉类食物如米饭和面食中,在蔬菜、水果、奶酪和肉类产品中也可分离到。此外,在植物根部、种子和昆虫的肠道也发现了产cereulide菌株^[28]。因此,产cereulide菌株的广泛流行,加之cereulide的稳定性质都对食品安全构成了严重威胁。

Cereulide通常引起恶心和呕吐的临床症状,症状一般在摄入cereulide污染的食物后0.5—6 h内出现,持续24 h后可自愈^{[19, 29]}。目前研究认为,cereulide通过与胃肠道内分布的5-HT₃受体结合,刺激迷走神经进而引发呕吐症状（图1-A）^[30]。10 μg·kg^-1的cereulide即可诱导恒河猴出现呕吐症状^[31],而人的诱导剂量目前尚不清楚。一般来说,cereulide常以中低浓度水平暴露于食物中,有研究发现在餐馆随机收集的米饭样本中,cereulide的检出率为7.4%,平均浓度为4 ng·g^{-1 [32]}。但也有食物中低剂量cereulide（0.01—1.28 ng·g^-1）导致食物中毒事件的报道,即体重70 kg的成年人摄入100 g此类食物便会出现呕吐症状^[33]。因此,低剂量的cereulide虽然不会立即引起呕吐反应,但由于容易被忽视导致消费者或反复暴露于亚致吐剂量,长期可能会对健康产生影响。

图1

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图1Cereulide的毒性及作用机制

A：Cereulide存在于多种食物中,通过与胃肠道内分布的5-HT₃受体结合,刺激迷走神经进而引发呕吐症状;B：Cereulide作为钾离子载体,会破坏细胞脂质膜上的电化学电位梯度,通过影响呼吸作用,最终导致细胞死亡
Fig. 1Toxicity and mechanism of cereulide

A: Cereulide is present in a variety of foods. It induces vomiting by binding to 5-HT₃ receptors in the gastrointestinal tract and stimulating the vagus nerve; B: Cereulide, as a potassium ionophore, disrupts the electrochemical potential gradient on lipid membranes which affects respiration, and ultimately leads to cell death


Cereulide还可导致人的急性肝功能衰竭、心脏衰竭和脑部水肿等症状,严重的可致人死亡^[4],但目前这些毒性作用机制都尚未研究清楚。当前对于cereulide的毒性作用机制研究多集中于细胞毒性作用,如研究表明cereulide对多种哺乳动物细胞具有损伤作用^[34]。cereulide可以使人喉癌细胞HEp2和人肝癌细胞HepG2细胞空泡化和线粒体损伤^[35,36];cereulide可导致人结肠腺癌细胞CaCo2、人肺腺癌细胞Calu3、宫颈癌细胞HeLa以及成神经细胞瘤细胞Paju的损伤^[37];还能抑制NK细胞,降低免疫力^[35];低浓度的cereulide会诱导胰岛β细胞凋亡,这与糖尿病的发生密不可分^{[34, 38]}。最近,研究报道了cereulide对Caco2和HepG2细胞中线粒体的影响,特别是在HepG2细胞系中,低剂量的cereulide可以完全抑制线粒体的呼吸作用（图1-B）^{[36, 39]}。

此外,本团队前期研究分析了cereulide在兔体内的毒代动力学规律,cereulide在体内的消除半衰期为（10.8±9.1）h^[40],消除半衰期短解释了cereulide引起短暂的呕吐症状并一般为自限性的临床特征,但其毒性的分子作用机制仍需进一步探索。

2 Cereulide的生物合成特征

NRPS合成系统是一种大型的模块化多酶复合物,参与多种天然产物的生物合成,如抗生素、表面活性剂和毒素^[41]。其生物合成途径可分为3类：线性（图2-A）,迭代型（图2-B）和非线性（图2-C）^[42],其中模块数量和顺序与产物肽中氨基酸的数量和顺序相匹配,并且各个模块通过催化反应与保守结构域协同作用,以定向方式将单体结合到天然产物肽中,从而确定最终产物的化学特性。其中腺苷酸化（adenylation, A）结构域,肽基载体蛋白（peptide carrier protein, PCP）或硫醇化（thiolation, T）结构域、缩合（Ccondensation, C）结构域以及硫酯酶（thioesterase, TE）结构域是NRPS合成的基本功能单元。合成过程一般为：首先,A域通过腺苷酸化作用特异性识别底物并激活合成前体;其次,PCP域负责携带不断增长的天然产物链在每个模块间穿梭,以此不断催化肽链延伸;C域一般位于模块末端,通过形成酰胺键或酯键催化链增长;最后,一旦非核糖体肽的合成完成,成熟的肽链将被转移到NRPS末端的TE域,通过水解释放线性肽,或通过分子内亲核攻击形成环状肽^[43]。

图2

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图2NRPS的3种生物合成途径

NRPS系统的A型生物合成途径可合成线性肽,例如枯草芽孢杆菌(B. subtilis)中的表面活性素(surfactin)。B型途径合成迭代肽,典型代表为蜡样芽孢杆菌(B. cereus)的致吐毒素(cereulide)。C型途径控制合成非线性肽,例如霍乱弧菌(Vibrio cholerae)的弧菌杆菌素(vibriobactin)
Fig. 2Three biosynthesis pathways of NRPSs

Type A biosynthesis pathway of NRPS system synthesizes linear peptides, such as surfactin in B. subtilis. Type B pathway synthesizes iterative peptides like cereulide in B. cereus. Type C pathway encodes nonlinear peptides, such as vibriobactin in Vibrio cholerae


Cereulide是NRPS迭代型合成途径的典型产物,由ces基因簇（cereulide synthetase gene cluster）控制合成^[44]。ces基因簇位于大小约270 kb的质粒上,该质粒与炭疽芽孢杆菌的毒力质粒pXO1具有高度相似性^[45]。ces基因簇包括cesH、cesP、cesT、cesA、cesB、cesC和cesD等7个主要编码区。cesA和cesB是典型的NRPS系列基因,负责cereulide肽链结构的组装;cesP是4'-磷酸泛酰巯基乙胺基转移酶基因,对于激活cereulide合成系统至关重要;cesT推测为II型硫酯酶基因,可以去除引发错误的单体;此外,cesH位于5'末端,推定为编码水解酶的基因;cesC/D位于3'端,推测为编码ABC转运蛋白的基因（图3-B）^[46,47]。

图3

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图3Cereulide的生物合成过程

A：Cereulide主要通过三个步骤完成生物合成;B：本文总结并提出的cereulide生物合成过程及各结构域功能
Fig. 3Biosynthetic pathway of cereulide

A: Cereulide is synthesized by three-step biosynthetic processes; B: Proposed biosynthetic pathway of cereulide and functions of each domain


ces基因簇的转录是在细菌对数生长晚期开始,并在转变至稳定期后迅速关闭^[48]。这种严格的时间调控是依赖于如AbrB、Spo0A和CodY等转录调节因子实现的。在蜡样芽孢杆菌中,AbrB和Spo0A调节子与芽孢的形成相关,而CodY的功能是调节细菌染色体与ces基因簇所在质粒间的信号传导,进一步将细胞的营养和能量状态与致病性联系起来^[49]。cereulide的合成除受细菌内部基因调控之外,还受所在环境因素的影响。例如,蜡样芽孢杆菌合成cereulide的最适温度为20—30 ℃,并且依赖氧气的存在,有研究表明在氧浓度低于1%—2%的条件下,菌株将不会产生cereulide^[28]。此外,营养条件、pH、湿度和NaCl等因素都会影响cereulide的产生^[50,51],但这些外部信号如何被细菌感知,转导并调节cereulide的合成过程这些问题均未阐明。

3 Cereulide的生物合成过程

随着近年来对NRPS合成天然产物的研究增多,cereulide的生物合成过程也得到了进一步探索。MAGARVEY等提出ces-NRPS识别的底物并不全是氨基酸,酮酸也是识别底物之一,例如CesA1的A结构域识别的是α-酮基羧酸（D-α-ketocarboxylic acids, KIC）,CesB1识别的底物为α-酮异戊酸（L-α- ketoisovaleric acid, KIV）,并提出了酮还原酶（KR）结构域的作用^[20]。此外,有研究提出了关于cereulide生物合成的新概念,即二肽是形成cereulide肽链的基础单元,而不是单个氨基酸或羟基酸作为组装的基本模块^[52]。并将CesA末端的缩合结构域描述为C^*域,该结构域的功能是酯合酶而不是酰胺合酶,推测其在cereulide肽链的最终环化过程中与TE结构域协同酯化作用^[21]。

在现有文献报道和研究数据的基础上,我们总结并提出了cereulide的生物合成机理,并着重探讨TE结构域和KR结构域在cereulide合成中的迭代和环化作用,如图3-A所示：

（1）二肽的合成：cereulide的主要合成单元,CesA由A-PCP-C-A-E-C^*结构域组成,CesB包含A-PCP-A- PCP-TE结构域。首先,CesA1,CesA2,CesB1和CesB2分别识别D-α-酮羧酸（KIC）,L-丙氨酸（Ala）,L-α-酮异戊酸（KIV）和L-缬氨酸（Val）。然后,通过KR域将α-KIC（CesA1）和α-KIV（CesB1）分别还原为D-α-羟基异己酸（HIC）和L-α-羟基异戊酸（HIV）。PCP结构域通过4'-磷酸泛酰巯基乙胺的巯基捕获活化的前体物质,C结构域催化下游PCP结合的单体,并对上游PCP结合的单体进行亲核攻击,最终共价结合形成二肽。

（2）四肽的合成：依照上述过程,CesA和CesB分别合成D-HIC-D-Ala和L-HIV-L-Val,C^*结构域催化酯键的形成,释放PCP上结合的四肽D-HIC-D- Ala-L-HIV-L-Val,该四肽随后转移至CesB末端TE结构域的丝氨酸羟基（Ser-OH）上。

（3）十二肽的环化：通过重复反应合成第二个四肽,两个四肽通过酯化形成八肽;再次重复上述反应,形成三元络合的产物肽。由于ces-NRPS的TE结构域活性中心表面结构阻止外部水分子进入,并诱导内部亲核攻击反应,最后TE结构域催化中间反应并释放环状的cereulide。

3.1 KR结构域的迭代作用-酯键的形成

Cereulide是十二环肽,由交替的6个酰胺键和6个酯键连接而成。ces-NRPS具有两个A结构域,其中在a8和a9之间插入了一个KR结构域（图3-B）^[20,53]。

KR的功能是利用NADPH作为辅助因子,催化α-酮酸还原为α-羟基酸,是酯键形成的基础^[52]。其他研究也证明了KR结构域的还原作用,例如在放线菌kutzerides合成中的KR结构域蛋白KtzG,以及链霉菌antimycins合成过程中的KR结构域蛋白AntC,均参与催化α-酮酸的还原^[54,55]。不同的是,kutzerides和antimycins合成基因簇中的KR结构域未插入A结构域中,而是位于A和PCP结构域之间并形成独立部分。此外,真菌的环二肽合成酶系统（cyclodepsipeptide synthetase systems）合成的如enniation和bassianolide,这些肽类同cereulide类似,也由酯键链接,但与细菌NRPS系统不同的是,真菌环二肽合成酶簇的A结构域可直接利用α-羟酸,无需KR结构域进行催化还原^[56]。总之,细菌NRPS系统的组成是灵活的,通过了解多肽类天然产物的迭代形成过程,可为后期通过基因工程、合成生物学和其他手段获得理想产物提供新的思路。

3.2 TE结构域的环化作用

许多NRPS合成的天然产物TE结构域的功能已得到良好阐述,其功能主要为：活性丝氨酸残基攻击PCP结构域结合的中间肽,并使之与TE结构域结合,成熟肽最终通过水解或环化作用从TE结构域释放,形成线性肽或环肽（图3-B）^[57,58]。例如,有研究分析了枯草芽孢杆菌surfactin的TE结构域晶体结构,SrfTE是一个含有碗状活性位点的球状结构域^[59]。并且与典型的α/β水解酶家族一样,SrfTE域也具有保守的Ser-His-Asp催化三联体^{[43, 60]}。因此,推测cereulide的TE结构域“肽结合袋”表面分布有大量疏水残基形成疏水环境,外部水分子被阻拦,分子内羟基或氨基对羰基碳发起亲核攻击,最终通过形成内酯或内酰胺释放环肽。

此外,也有研究表明若TE结构域活性中心位点上氨基酸为结构稳定、刚性的脯氨酸时,则会形成环状产物;当脯氨酸被骨架柔软的氨基酸替代时,外部水分子进入TE结合口袋充当亲核试剂,则会形成线性肽^[58]。总之,阻止外部水分子进入TE的“肽结合袋”,是形成环状产物的关键。另一方面,我们认为TE结构域也具有调节迭代次数的功能。例如,enniation也含有3个重复单元,其C末端的TE结构域被PCP-C结构域取代,这表明TE结构域和PCP-C结构域可能具有相似的功能或核心位点,而PCP域的功能便是控制天然产物链的延伸长度^[43]。

4 结语

综述了蜡样芽孢杆菌主要毒素cereulide的毒性作用和生物合成过程,其毒性作用机制主要包括：与5-HT₃受体结合,刺激传入迷走神经引起呕吐;诱导线粒体膜电位耗竭,导致细胞死亡。然而,cereulide导致的急性肝衰竭、心力衰竭、横纹肌溶解和脑部病变等毒性作用的机制尚不清楚。此外,在综述cereulide的生物合成过程中,笔者强调了催化酮酸形成酯的酮还原酶（KR）域,及形成重复单元和环肽的硫酯酶（TE）域在cereulide合成过程中的迭代和环化作用。并且这种作用并不仅限于cereulide,而是可以作为NRPS系统生物合成环状迭代天然产物的推广分析,为后期通过基因工程、合成生物学和其他手段获得理想产物等研究提供科学依据。

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