本文選題：棘霉素 + 喹喔啉環(huán)�。� 參考：《上海交通大學(xué)》2013年博士論文

【摘要】：棘霉素(echinomycin)是具有良好的抗腫瘤活性的非核糖體肽(NRP)類的天然產(chǎn)物，其結(jié)構(gòu)除了有非核糖體肽合酶（NRPS）合成的環(huán)肽骨架外，還具有兩個(gè)喹喔啉環(huán)生色團(tuán)。棘霉素正是通過這兩個(gè)扁平狀的喹喔啉環(huán)插入到DNA分子的堿基對(duì)之間，從而抑制了DNA的復(fù)制和轉(zhuǎn)錄。正是因?yàn)檫@個(gè)性質(zhì)，該化合物作為抗腫瘤藥物而被開發(fā)。棘霉素對(duì)DNA的結(jié)合具有一定的偏好性，其插入熱點(diǎn)通常是側(cè)翼為AT堿基對(duì)，中間為5′-CG-3′的序列，這是由棘霉素的環(huán)肽骨架中的氨基酸殘基與DNA中的堿基通過非共價(jià)鍵進(jìn)行特異的相互作用所決定的。同位素喂養(yǎng)實(shí)驗(yàn)發(fā)現(xiàn)其中的喹喔啉環(huán)的生物合成是以L-色氨酸為前體，此外還確定了(2S，3S) β-羥基色氨酸和β-羥基犬尿氨酸兩個(gè)中間產(chǎn)物。但具體的生物合成途徑一直都在研究和論證中。本課題出發(fā)菌株是由廣東省農(nóng)科院篩選的灰色變異鏈霉菌萬隆亞種，它是通過太空誘變育種得到的突變株，也是擁有我國自主知識(shí)產(chǎn)權(quán)的微生物資源。 利用棘霉素產(chǎn)生菌S. lasaliensis和S. triostinicus中的棘霉素生物合成基因簇中的喹喔啉-2-甲酸(QXC)腺苷�；富騟cm1和trsA為探針，從構(gòu)建的灰色變異鏈霉菌萬隆亞種Streptomycesgriseovariabilis subsp. bandungensis subsp. nov基因組文庫中篩選到2個(gè)陽性fosmidA111和K311。通過構(gòu)建亞克隆和序列分析，發(fā)現(xiàn)K311含有所有A311中包含的棘霉素生物合成的相關(guān)基因�；疑儺愭溍咕f隆亞種中棘霉素生物合成基因簇與S. triostinicus的棘霉素生物合成基因簇具有較高同源性，并且具有相同基因排布。與S. lasaliensis棘霉素生物合成基因簇相比，具有較低同源性和完全不同的基因排布。結(jié)合生物信息學(xué)分析，預(yù)測(cè)出灰色變異鏈霉菌萬隆亞種中含有18個(gè)基因的棘霉素生物合成基因簇，包括3個(gè)與NRP骨架合成相關(guān)基因qui6、qui7、qui16，7個(gè)與喹喔啉環(huán)生物合成相關(guān)基因qui3、qui12、qui13、qui14、qui15、qui17、qui18，2個(gè)NRP骨架后修飾基因qui8、qui11，3個(gè)抗性基因qui1、qui2、qui10，1個(gè)調(diào)控基因qui4和2個(gè)未知功能基因qui5、qui9。在此基礎(chǔ)上通過對(duì)關(guān)鍵基因的功能研究，解析了棘霉素芳香族結(jié)構(gòu)單元喹喔啉環(huán)的生物合成途徑。 根據(jù)Kenji Watanabe的假說，喹喔啉環(huán)的生物合成在最初的階段經(jīng)歷了從L-色氨酸直至N-甲酰-β-羥基犬尿氨酸的轉(zhuǎn)化。然而，該假說完全基于氘標(biāo)記的(2S,3S) β-羥基色氨酸和(2S,3R)β-羥基犬尿氨酸喂養(yǎng)實(shí)驗(yàn)，因此有必要對(duì)催化上訴幾步反應(yīng)的酶分別進(jìn)行體外研究來檢驗(yàn)特定的底物是否能產(chǎn)生預(yù)期的產(chǎn)物。最終，，我們對(duì)類MbtH蛋白Qui5、雙結(jié)構(gòu)域NRPS蛋白Qui18、依賴于細(xì)胞色素P450的羥化酶Qui15和色氨酸2，3-雙加氧酶Qui17的體外功能驗(yàn)證肯定了上述步驟，并揭示了其中每個(gè)酶生化功能。 首先，在對(duì)雙結(jié)構(gòu)域NRPS蛋白Qui18的研究中，我們發(fā)現(xiàn)類MbtH蛋白Qui5對(duì)Qui18的輔助作用是L-色氨酸加載到Qui18的PCP結(jié)構(gòu)域上所必需的。起初，我們發(fā)現(xiàn)單獨(dú)表達(dá)的Qui18溶解性很差，當(dāng)我們構(gòu)建了一個(gè)pET28-a的衍生型表達(dá)載體pCT28，并用它把qui5和qui18串聯(lián)在一起，這樣共表達(dá)出來的Qui18溶解性提高了近100倍，對(duì)這個(gè)共表達(dá)產(chǎn)物進(jìn)行分子篩分析發(fā)現(xiàn)，兩個(gè)Qui5和兩個(gè)Qui18形成四聚體，并且只有用共表達(dá)的蛋白，才能檢測(cè)到L-色氨酸的加載。用單獨(dú)表達(dá)的Qui18，或是在單獨(dú)表達(dá)的Qui18中補(bǔ)加Qui5都不能檢測(cè)到色氨酸的加載。 其次，確定了加載的L-色氨酸能被依賴于細(xì)胞色素P450的羥化酶Qui15羥化，而該酶對(duì)游離的L-色氨酸不起作用。在對(duì)依賴于細(xì)胞色素P450的羥化酶Qui15研究中發(fā)現(xiàn)，它只能催化加載到Qui18上的色氨酸，使其β碳加上羥基。而對(duì)游離的L-色氨酸不起作用。 最后，通過體外酶催化分析，確定了色氨酸雙加氧酶Qui17能催化（2S,3S）β-羥基色氨酸，生成N-甲酰-β-羥基犬尿氨酸。由于Qui15所催化的羥化反應(yīng)生成的產(chǎn)物量有限，并且還需要進(jìn)行水解才能將β-羥基色氨酸釋放出來，因此如此少量的β-羥基色氨酸很難滿足研究色氨酸2，3-雙加氧酶Qui17對(duì)底物的需求。于是，為了在體外研究Qui17，我們按照Kenji Watanabe的方法，用化學(xué)手段合成了(2S,3S) β-羥基色氨酸。我們發(fā)現(xiàn)Qui17與一般的色氨酸雙加氧酶不同，一般的色氨酸雙加氧酶只能催化L-色氨酸和5-氟色氨酸，而Qui17確實(shí)可以催化(2S,3S) β-羥基色氨酸，生成N-甲酰-β-羥基犬尿氨酸。對(duì)Qui17的空間結(jié)構(gòu)進(jìn)行了同源建模，嘗試性分析了與羥基色氨酸的羥基有特異結(jié)合的氨基酸殘基情況。 通過以上喹喔啉環(huán)生物合成的前幾步反應(yīng)的驗(yàn)證，灰色變異鏈霉菌萬隆亞種中棘霉素生物合成途徑逐漸明確，但接下來的反應(yīng)直至喹喔啉環(huán)的生成仍然是個(gè)不確定的謎，這也是我們將要進(jìn)行的工作。既然喹喔啉環(huán)對(duì)棘霉素的活性有著至關(guān)重要的作用，研究喹喔啉環(huán)的生物合成途徑會(huì)為通過組合生物合成手段進(jìn)行醌霉素類天然產(chǎn)物結(jié)構(gòu)改造，獲得更有效的醌霉素類似物做出貢獻(xiàn)。
[Abstract]:Acanthomycin (echinomycin) is a natural product of non ribosomal peptide (NRP), which has good antitumor activity. Besides the cycP skeleton synthesized by non ribosomal peptide synthase (NRPS), it also has two cycic chromophores, which are inserted between the base pairs of the DNA molecule by these two flat flat ooazine rings. It inhibits the replication and transcription of DNA. It is precisely because of this nature that the compound has been developed as an antitumor drug. Acanthomycin has a certain preference for the binding of DNA, and its insertion hot spots are usually flanking to AT base pairs, with a sequence of 5 '-CG-3' in the middle, which is the amino acid residues in the cytoskeleton skeleton of acanthomycin and the alkali in DNA. It was determined by the specific interaction of non covalent bonds. The isotopic feeding experiments found that the biosynthesis of the L- tryptophone ring was the precursor of tryptophan, in addition to the two intermediate products of (2S, 3S) beta hydroxytryptophan and beta hydroxy canine urinary ammonia, but the specific biosynthetic pathway was always studied and demonstrated. The strain of this project is a strain of Streptomyces mutants, selected from the Academy of Agricultural Sciences in Guangdong Province, which is a mutant strain obtained through space mutation breeding, and also a microbial resource with independent intellectual property rights in China.
S. lasaliensis and QXC adenosine acylase gene ECM1 and trsA in the acanthmycin biosynthesis gene cluster of the acanthomycin producing bacteria S. lasaliensis and S. triostinicus were selected as the probes, and 2 were screened from the constructed Streptomycesgriseovariabilis subsp. bandungensis subsp. genomic library of the subspecies of Streptomyces mutant. The positive fosmidA111 and K311. found that K311 contained all the genes involved in the biosynthesis of acanthomycin in all A311 by constructing the subclone and sequence analysis. The genetic cluster of acanthomycin biosynthesis gene cluster in the subspecies of Streptomyces gray Streptomyces and S. triostinicus has high homology and has the same gene. Arrangement. Compared with the S. lasaliensis anthomycin biosynthesis gene cluster, it has low homology and completely different gene arrangement. Combined with bioinformatics analysis, it predicts the acanthomycin biosynthesis gene cluster containing 18 genes in the subspecies of Streptomyces grey Streptomyces, including 3 qui6, qui7, and qui16,7 related genes related to NRP skeleton synthesis. Qui3, qui12, qui13, qui14, qui15, qui17, qui18,2 NRP skeleton modified gene qui8, qui11,3 resistant gene qui1, qui2, regulatory genes and 2 unknown functional genes, based on the study of the function of the key genes, the aromatic structural units of the acanthomycin were analyzed. Biosynthesis pathway of the Al - Q ring.
According to the hypothesis of Kenji Watanabe, the biosynthesis of the olinine ring has experienced the transformation from L- tryptophan to N- formyl - beta hydroxy canine urinary ammonia. However, the hypothesis is entirely based on the deuterium labeled (2S, 3S) beta hydroxytryptophan and (2S, 3R) beta hydroxy canine urinary ammonia feeding experiments, so it is necessary to respond to a few steps of catalytic appeals. The enzyme was studied in vitro to test whether specific substrates could produce expected products. Finally, we affirmed the above steps for the functions of MbtH protein Qui5, double domain NRPS protein Qui18, cytochrome P450 hydroxylase Qui15 and tryptophan 2,3- bioxygenase Qui17 in vitro, and revealed the biochemical work of each enzyme. Yes.
First, in the study of the double domain NRPS protein Qui18, we found that the auxiliary effect of the MbtH protein Qui5 on Qui18 was necessary for L- tryptophan loading to the PCP domain of Qui18. At first, we found that the solo solubility of Qui18 was very poor, when we constructed a pET28-a derivative that expressed the carrier pCT28, and used it for qui5 and Qui18 was linked together, so the co expressed Qui18 solubility was increased by nearly 100 times. Molecular sieve analysis of the co expression product found that two Qui5 and two Qui18 formed four polymers, and only a co expressed protein could be used to detect the loading of L- tryptophan. Qui18, individually expressed, or supplemented in a separate Qui18. The loading of tryptophan can not be detected with Qui5.
Secondly, it was determined that the loaded L- tryptophan was able to be hydroxylated by the hydroxylase Qui15 dependent on cytochrome P450, which did not act on free L- tryptophan. In the study of the hydroxylase Qui15 dependent on the cytochrome P450, it could only catalyze the loading of tryptophan on Qui18 to make its beta carbon add hydroxyl group and to free L- tryptophan. Work.
Finally, the tryptophan dioxygenase Qui17 can catalyze (2S, 3S) beta hydroxytryptophan to produce N- formyl - beta hydroxyl tryptophan. The production of the hydroxylation of Qui15 catalyzed by Qui15 is limited, and it needs to be hydrolyzed to release beta hydroxytryptophan and so a small amount of beta hydroxyl. Tryptophan is difficult to meet the need to study the substrate of tryptophan 2,3- dioxygenase Qui17. So, in order to study Qui17 in vitro, we synthesized (2S, 3S) beta hydroxytryptophan by chemical method in accordance with Kenji Watanabe method. We found that Qui17 is different from the normal tryptophan dioxygenase, and the general tryptophan dioxygenase can only catalyze L-. Tryptophan and 5- fluorotryptophan, and Qui17 really can catalyze (2S, 3S) beta hydroxytryptophan and produce N- - beta hydroxy canine urinary ammonia. The spatial structure of Qui17 is modeled and the amino acid residues with specific binding to hydroxyl hydroxyl groups of hydroxyl tryptophan are analyzed.
The biosynthesis pathway of acanthomycin in the subspecies of Streptomyces mutant was gradually clear by the previous reaction of the biosynthesis of the biosynthesis of the above, but the subsequent reaction until the formation of the Q ring is still an uncertain mystery, and this is the work we are going to do. Since the activity of the azo ring to the acanthomycin The study of biosynthesis pathway of quinazine rings will contribute to the improvement of natural product structure of quinamycin by combined biosynthesis, and more effective quinamycin analogues.
【學(xué)位授予單位】：上海交通大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2013
【分類號(hào)】：Q93

【共引文獻(xiàn)】

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