棘霉素中喹喔啉环的生物合成途径研究

发布时间：2018-05-17 01:17

本文选题：棘霉素 + 喹喔啉环　；参考：《上海交通大学》2013年博士论文

【摘要】：棘霉素(echinomycin)是具有良好的抗肿瘤活性的非核糖体肽(NRP)类的天然产物，其结构除了有非核糖体肽合酶（NRPS）合成的环肽骨架外，还具有两个喹喔啉环生色团。棘霉素正是通过这两个扁平状的喹喔啉环插入到DNA分子的碱基对之间，从而抑制了DNA的复制和转录。正是因为这个性质，该化合物作为抗肿瘤药物而被开发。棘霉素对DNA的结合具有一定的偏好性，其插入热点通常是侧翼为AT碱基对，中间为5′-CG-3′的序列，这是由棘霉素的环肽骨架中的氨基酸残基与DNA中的碱基通过非共价键进行特异的相互作用所决定的。同位素喂养实验发现其中的喹喔啉环的生物合成是以L-色氨酸为前体，此外还确定了(2S，3S) β-羟基色氨酸和β-羟基犬尿氨酸两个中间产物。但具体的生物合成途径一直都在研究和论证中。本课题出发菌株是由广东省农科院筛选的灰色变异链霉菌万隆亚种，它是通过太空诱变育种得到的突变株，也是拥有我国自主知识产权的微生物资源。利用棘霉素产生菌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)腺苷酰化酶基因ecm1和trsA为探针，从构建的灰色变异链霉菌万隆亚种Streptomycesgriseovariabilis subsp. bandungensis subsp. nov基因组文库中筛选到2个阳性fosmidA111和K311。通过构建亚克隆和序列分析，发现K311含有所有A311中包含的棘霉素生物合成的相关基因。灰色变异链霉菌万隆亚种中棘霉素生物合成基因簇与S. triostinicus的棘霉素生物合成基因簇具有较高同源性，并且具有相同基因排布。与S. lasaliensis棘霉素生物合成基因簇相比，具有较低同源性和完全不同的基因排布。结合生物信息学分析，预测出灰色变异链霉菌万隆亚种中含有18个基因的棘霉素生物合成基因簇，包括3个与NRP骨架合成相关基因qui6、qui7、qui16，7个与喹喔啉环生物合成相关基因qui3、qui12、qui13、qui14、qui15、qui17、qui18，2个NRP骨架后修饰基因qui8、qui11，3个抗性基因qui1、qui2、qui10，1个调控基因qui4和2个未知功能基因qui5、qui9。在此基础上通过对关键基因的功能研究，解析了棘霉素芳香族结构单元喹喔啉环的生物合成途径。根据Kenji Watanabe的假说，喹喔啉环的生物合成在最初的阶段经历了从L-色氨酸直至N-甲酰-β-羟基犬尿氨酸的转化。然而，该假说完全基于氘标记的(2S,3S) β-羟基色氨酸和(2S,3R)β-羟基犬尿氨酸喂养实验，因此有必要对催化上诉几步反应的酶分别进行体外研究来检验特定的底物是否能产生预期的产物。最终，，我们对类MbtH蛋白Qui5、双结构域NRPS蛋白Qui18、依赖于细胞色素P450的羟化酶Qui15和色氨酸2，3-双加氧酶Qui17的体外功能验证肯定了上述步骤，并揭示了其中每个酶生化功能。首先，在对双结构域NRPS蛋白Qui18的研究中，我们发现类MbtH蛋白Qui5对Qui18的辅助作用是L-色氨酸加载到Qui18的PCP结构域上所必需的。起初，我们发现单独表达的Qui18溶解性很差，当我们构建了一个pET28-a的衍生型表达载体pCT28，并用它把qui5和qui18串联在一起，这样共表达出来的Qui18溶解性提高了近100倍，对这个共表达产物进行分子筛分析发现，两个Qui5和两个Qui18形成四聚体，并且只有用共表达的蛋白，才能检测到L-色氨酸的加载。用单独表达的Qui18，或是在单独表达的Qui18中补加Qui5都不能检测到色氨酸的加载。其次，确定了加载的L-色氨酸能被依赖于细胞色素P450的羟化酶Qui15羟化，而该酶对游离的L-色氨酸不起作用。在对依赖于细胞色素P450的羟化酶Qui15研究中发现，它只能催化加载到Qui18上的色氨酸，使其β碳加上羟基。而对游离的L-色氨酸不起作用。最后，通过体外酶催化分析，确定了色氨酸双加氧酶Qui17能催化（2S,3S）β-羟基色氨酸，生成N-甲酰-β-羟基犬尿氨酸。由于Qui15所催化的羟化反应生成的产物量有限，并且还需要进行水解才能将β-羟基色氨酸释放出来，因此如此少量的β-羟基色氨酸很难满足研究色氨酸2，3-双加氧酶Qui17对底物的需求。于是，为了在体外研究Qui17，我们按照Kenji Watanabe的方法，用化学手段合成了(2S,3S) β-羟基色氨酸。我们发现Qui17与一般的色氨酸双加氧酶不同，一般的色氨酸双加氧酶只能催化L-色氨酸和5-氟色氨酸，而Qui17确实可以催化(2S,3S) β-羟基色氨酸，生成N-甲酰-β-羟基犬尿氨酸。对Qui17的空间结构进行了同源建模，尝试性分析了与羟基色氨酸的羟基有特异结合的氨基酸残基情况。通过以上喹喔啉环生物合成的前几步反应的验证，灰色变异链霉菌万隆亚种中棘霉素生物合成途径逐渐明确，但接下来的反应直至喹喔啉环的生成仍然是个不确定的谜，这也是我们将要进行的工作。既然喹喔啉环对棘霉素的活性有着至关重要的作用，研究喹喔啉环的生物合成途径会为通过组合生物合成手段进行醌霉素类天然产物结构改造，获得更有效的醌霉素类似物做出贡献。
[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.
【学位授予单位】：上海交通大学
【学位级别】：博士
【学位授予年份】：2013
【分类号】：Q93

【共引文献】