Ergot Alkaloid Biosynthesis in Aspergillus fumigatus: FgaAT Catalyses the Acetylation of Fumigaclavine B
Ergot Alkaloid Biosynthesis in Aspergillus fumigatus: FgaAT Catalyses the Acetylation of Fumigaclavine B
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烟曲霉中麦角生物碱的生物合成：FgaAT 催化烟曲霉 B 的乙酰化
DOI：
10.1002/cbic.200900395
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发表时间：
2009-09-21
期刊：
影响因子：
3.2
通讯作者：
Li, Shu-Ming
中科院分区：
文献类型：
--
作者：
Liu, Xiaoqing;Wang, Lu;Li, Shu-Ming
麦角生物碱是一类复杂的吲哚类化合物，主要由麦角菌属、青霉属和曲霉属真菌产生，具有多种生物活性和药理活性。棒麦角碱型麦角生物碱，例如烟曲霉产生的农棒麦角碱、烟曲霉碱A、B和C（方案1），仅由四环麦角林环组成。2]这些化合物具有与d-麦角酸相同的骨架和生物合成来源，所述d-麦角酸是药学上重要的麦角肽类（例如麦角胺）的酸性组分，来自麦角菌（Claviceps purpurea）（方案1）。许多麦角生物碱，包括A. fumigatus和ergopeptines; purpurea，而在这两种真菌的途径中的后续步骤不同（方案1）。麦角生物碱的生物合成是麦角菌次生代谢研究的重要领域，主要在麦角菌中进行。直到在C. 1999年发表的关于麦角生物碱的研究中，大多数是用麦角生物碱生产者的粗酶提取物进行的饲养试验和生化研究。通过使用dmaW基因进行基因组步移发现了该簇，该基因编码第一种途径特异性酶，4-二甲基烯丙基色氨酸合酶（4-DMATS）。该簇的鉴定为生物合成酶结构基因的分子生物学和生物化学研究提供了前提。6年后，利用生物信息学方法，在烟曲霉基因组序列中发现了烟曲霉素C生物合成的基因簇。烟曲霉Af 293。8]通过比较C中的两个簇，鉴定缺少肽基部分的棒型麦角生物碱的这个簇提供了鉴定参与麦角生物碱生物合成的共同阶段的候选基因的方便方法。purpurea和A.烟熏。通过鉴定二甲基烯丙基色氨酸N-甲基转移酶FgaMT，清楚地证明了这种策略的成功应用。FgaMT与数据库中的已知蛋白质没有显著的序列相似性，并且在两个簇中均被发现;这表明它在生物合成的早期阶段，即l-色氨酸和农麦角碱之间的作用。事实上，通过基因克隆和生物化学表征，可以表明FgaMT催化生物合成的第二步（方案1）。两个基因簇的比较也有助于发现参与生物合成后期步骤的候选基因，即在农棒霉素之后。这种方法通过在A.烟熏。fgaPT 1基因是烟曲霉素C基因簇中的第二个异戊二烯基转移酶。fumigatus，而C.紫花。这一发现很好地对应于两种菌株中麦角生物碱的结构差异之一，即在烟曲霉素C的C2位存在反向异戊二烯基部分，而在麦角肽类（如ACHTUNGTRENNUNGergotamine）中不存在该基团（方案1）。因此，该基因很可能参与烟曲霉素A向C的转化，这在生物化学上用过量生产和纯化的FgaPT 1证明（方案1）。同样的策略现在被用于鉴定和鉴定催化烟麦角碱B转化为A的乙酰转移酶。该反应仅在A.对C.这是因为在烟麦角碱A和C的结构中发现了乙酰基，而在麦角碱C的结构中未发现乙酰基。紫花。因此，编码乙酰基转移酶的基因应该只存在于烟曲霉素C的基因簇中。烟熏。两个基因簇在A.和C. purpurea中烟曲霉素C生物合成基因簇中的一个基因在A.烟曲霉AFUA_2G18020可能是该反应的候选菌株。使用FGENESH分析（参见支持信息）显示AFUA_2G18020仅由一个1485 bp的外显子组成。它跨越AAHF 01000001.1（GenBank）的碱基291 8826-292 0310。AFUA_2G18020的推导产物EAL 94101（在本研究中称为FgaAT）包含494个氨基酸，并且具有55.0kDa的计算分子量。数据库检索显示，FgaAT与真菌中的O-乙酰基转移酶具有较低的序列相似性，这些酶参与了倍半萜类真菌毒素的生物合成。例如，通过使用程序“BLAST 2 SEQUENCES”（参见支持信息），发现FgaAT与来自尖孢镰刀菌（Fusarium oxysporum）的二孢噻吩3- 0-乙酰基转移酶BAC 65220具有22%的同一性。这种相似性显然太低，不能将FgaAT识别为乙酰转移酶。然而，在烟曲霉ACHTUNGTRENNUNGvine C的生物合成基因簇中没有进一步的基因显示出与乙酰转移酶的相似性。因此，我们推测FgaAT将催化烟麦角碱B的上述乙酰化。[a]X博士教授柳湖，加-地首都师范大学生命科学学院北京西三环北路105号[B]。Wang，N. Steffan，W.- B。Yin，Prof. Dr. S.- M. Li Philipps-Universit t马尔堡，Institut f r Pharmazeutische Biologie Deutschhausstrasse 17 A，35037马尔堡（德国）传真：（+ 49）6421- shuming.li @ staff.uni-marburg.de本文的支持信息可在万维网http：//www.example.com下获得。dx.doi.org/10.1002/cbic.200900395
Ergot alkaloids are a complex family of indole derivatives with diverse biological and pharmacological activities and are produced mainly by fungi of the genera Claviceps, Penicillium and Aspergillus. Ergot alkaloids of the clavine-type, for example, agroclavine, fumigaclavines A, B and C produced by Aspergillus fumigatus (Scheme 1), consist merely of the tetracyclic ergoline ring. 2] These compounds have the same skeleton and biosynthetic origin as d-lysergic acid, the acidic component of the pharmaceutically important ergopeptines, for example, ergotamine, from Claviceps purpurea (Scheme 1). The early stages of the biosynthetic pathway up to agroclavine are very likely shared by many ergot alkaloids, including fumigaclavines in A. fumigatus and ergopeptines in C. purpurea, whereas the later steps in the pathways differ in these two fungi (Scheme 1). Investigations on the biosynthesis of ergot alkaloids, mainly carried out in Claviceps, have been an important research field in secondary metabolism. Until the identification of a biosynthetic gene cluster of ergot alkaloids in C. purpurea in 1999, most of these studies were feeding experiments and biochemical studies with crude enzyme extracts from ergot alkaloid producers. The cluster was found by genomic walking by using the dmaW gene, which codes for the first pathway-specific enzyme, a 4-dimethylallyltryptophan synthase (4-DMATS). The identification of this cluster provided the prerequisite for molecular biological and biochemical investigations on structure genes of biosynthetic enzymes. Six years later, a gene cluster for the biosynthesis of fumigaclavine C was identified with bioinformatics approaches in the genome sequence of A. fumigatus Af293. 8] The identification of this cluster for a clavinetype ergot alkaloid lacking the peptidyl moiety provided a convenient way to identify candidate genes that are involved in the common stages of the biosynthesis of ergot alkaloids by comparison of the two clusters in C. purpurea and A. fumigatus. Successful application of this strategy was clearly demonstrated by the identification of the dimethylallyltryptophan N-methyltransferase, FgaMT. FgaMT shares no significant sequence similarity with known proteins in databases and was found in both clusters ; this indicates its role in the early stage of biosynthesis, that is, between l-tryptophan and agroclavine. Indeed, by gene cloning and biochemical characterisation it could be shown that FgaMT catalysed the second step of the biosynthesis (Scheme 1). Comparison of both gene clusters also helps to find candidate genes that are involved in the later steps of the biosynthesis, that is, after agroclavine. This approach was demonstrated by the identification of the fumigacalvine A prenyltransferase FgaPT1 in A. fumigatus. The fgaPT1 gene was identified as a second putative prenyltransferase in the gene cluster of fumigaclavine C in A. fumigatus, but not in C. purpurea. This finding corresponded well to one of the structural differences of the ergot alkaloids in the two strains, that is, the presence of a reverse prenyl moiety at position C2 of fumigaclavine C and absence of this group in ergopeptines, like ACHTUNGTRENNUNGergotamine (Scheme 1). Therefore, this gene is very likely involved in the conversion of fumigaclavine A to C, which was proven biochemically with overproduced and purified FgaPT1 (Scheme 1). The same strategy was now used to identify and characterise the acetyltransferase that catalyses the conversion of fumigaclavine B to A. This reaction is only expected in the biosynthesis of fumigaclavine C in A. fumigatus, but not of ergot alkaloids in C. purpurea, because an acetyl moiety was only found in the structure of fumigaclavine A and C, but not in the structures of ergot alkaloids in C. purpurea. Therefore, the gene coding the expected acetyltransferase should be exclusively found in the gene cluster of fumigaclavine C in A. fumigatus. Comparison of both gene clusters in A. fumigatus and C. purpurea indicated that one gene in the biosynthetic gene cluster of fumigaclavine C in A. fumigatus, that is, AFUA_2G18020, could be the candidate for this reaction. Analysis by using FGENESH (see the Supporting Information) revealed that AFUA_ 2G18020 consists only of one exon of 1485 bp. It spans bases 291 8826–292 0310 of AAHF01000001.1 (GenBank). The deduced product of AFUA_2G18020, EAL94101 (termed FgaAT in this study) comprises 494 amino acids and has a calculated molecular mass of 55.0 kDa. Database search showed that FgaAT shares low sequence similarity with O-acetyltransferases from fungi, which are involved in the biosynthesis of trichothecenes, a group of sesquiterpenoid mycotoxins. For example, by using the program “BLAST 2 SEQUENCES” (see the Supporting Information) FgaAT was found to share an identity of 22 % with trichothecene 3-O-acetyltransferase BAC65220 from Fusarium oxysporum. This similarity is obviously too low for FgaAT to be recognised as an acetyltransferase. However, no further genes in the biosynthetic gene cluster of fumigaclaACHTUNGTRENNUNGvine C shows a similarity to acetyltransferases. Therefore, we speculated that FgaAT would catalyse the mentioned acetylation of fumigaclavine B. [a] Prof. Dr. X. Liu, L. Wang College of Life Science, Capital Normal University No. 105 Xisanhuan Beilu, Beijing, 100048 (China) [b] L. Wang, N. Steffan, W.-B. Yin, Prof. Dr. S.-M. Li Philipps-Universit t Marburg, Institut f r Pharmazeutische Biologie Deutschhausstrasse 17 A, 35037 Marburg (Germany) Fax: (+ 49) 6421-2825365 E-mail : shuming.li@staff.uni-marburg.de Supporting information for this article is available on the WWW under http ://dx.doi.org/10.1002/cbic.200900395.