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Thesis Advisor郑舰艇
Degree Grantor中国科学院研究生院
Place of Conferral北京
Degree Discipline生物工程
Keyword热稳定酰基coa合成酶 生化特性分析 底物特异性分析 定点突变

聚酮化合物是由细菌、真菌、植物等产生的次级代谢产物,具有多种生物活性,广泛应用于人类生产生活中。聚酮结构一般较为复杂且常具有多个手性中心,化学合成困难,目前主要通过微生物发酵获得。阐明聚酮生物合成机制,是聚酮化合物研究的重要内容。酰基辅酶A(coenzyme A,CoA)如丙酰CoA、乙酰CoA、丙二酰CoA以及甲基丙二酰CoA等是聚酮生物合成的前体,但商品化产品种类较少且往往价格昂贵,严重限制聚酮生物合成的体外研究。酰基CoA合成酶可以催化羧酸与CoA缩合,为聚酮的生物合成提供前体,是聚酮研究的重要工具酶。热稳定性酶的适用范围广且纯化简单,是理想的工具酶,但已报道的酰基CoA合成酶多为来源于常温菌的中温酶,一般不具备热稳定性。本论文异源表达了来源于嗜热菌Caldicellulosiruptor Owensensis OL的热稳定酰基CoA合成酶CoACS,体外生化实验证明该酶有较好的底物宽泛性及较高的催化活性,是提供聚酮合成前体的理想工具酶。论文的主要内容包括:首先,通过序列比对、保守模体(motif)分析及分子进化树构建的方法在嗜热菌C. Owensensis OL基因组中找到编码酰基CoA合成酶的基因Calow_0635,成功构建了表达载体,并实现了在大肠杆菌中的过量表达。经过Ni柱亲和层析及透析得到目标蛋白CoACS。其次,探究了CoACS的底物特异性及生化特性。用乙酸、丙酸、丙二酸等十种羧酸考察了CoACS的底物特异性,发现CoACS能够催化丙酸、丁酸、2-甲基丙酸、戊酸、2-甲基丁酸、3-甲基丁酸、环己甲酸这七种单羧酸,但不具备催化乙酸、丙二酸和2,3-二甲基丙烯酸的能力。依次测定CoACS对七种不同单羧酸的催化比活力,结果显示CoACS对于戊酸和丁酸的催化活性较好,而对于带有支链的2-甲基丙酸、2-甲基丁酸、3-甲基丁酸的催化活性则较低。以催化活性最高的戊酸作为底物,考察CoACS的最适反应条件,发现其在30 ℃,pH 7.0的条件下活性最高。热稳定性实验表明该酶在70 ℃的高温下温育8小时后仍保留有45%的活性。酶动力学参数测定结果显示CoACS对戊酸的kcat/Km值为783.56 M-1s-1,远高于其它已报道的酰基CoA合成酶。最后,分析了CoACS不能催化丙二酸和2,3-二甲基丙烯酸的原因,并通过定点突变实现了CoACS底物特异性的转变。经过对丙二酰CoA合成酶的结构及序列分析发现它们的序列中存在三个保守氨基酸而CoACS不具备,推测酰基CoA合成酶识别丙二酸与这三个氨基酸相关。将CoACS中相应的三个氨基酸定点突变(F243H、A314S、A336R)后,考察底物特异性发现其具备了催化丙二酸的活性,但失去了催化戊酸的能力。将2,3-二甲基丙烯酸对接进能催化2,3-二甲基丙烯酸的苯甲酰CoA合成酶(pdb:4EAT)的蛋白结构中,找出了稳定底物2,3-二甲基丙烯酸分子的关键氨基酸。

Other Abstract

Polyketides, a class of secondary metabolites produced by bacterium, fungi and plants, have many kinds of bioactivities and play an important role in human life and production. With complex structures and chiral atoms, polyketides are difficult to synthesize through chemical method, they are mainly acquired by microbial fermentation at present. As a consequence, the vital part of researches on polyketides is to understand the mechanism of polyketides biosynthesis. Acyl Coenzyme A (CoA) such as acetyl-CoA, propionyl-CoA, malonyl-CoA and methylmalonyl-CoA, are common precursors selected by polyketide synthetases. However, sorts of acyl CoA which can be obtained from commercial source are limited and the prices of them are high. The difficulty of access to these precursors has prevented studies on the polyketides synthesis in vitro. Acyl CoA synthetases, which can provide building blocks for polyketide biosynthesis, are useful tool enzymes for the study of polyketide synthesis. Thermostable enzymes are popular as tool enzymes because of their wide adaptability and simple purification. Nevertheless, acyl CoA synthetases which have been reported usually come from mesophilic bacterial and do not have thermostablity. In this study, one thermostable acyl CoA synthetase (CoACS) which was form thermophilic bacteria Caldicellulosiruptor Owensensis OL was heterologous expressed and characterized. Results demonstrated that CoACS had wide substrate specificity and high catalytic activity and could be used as an excellent tool enzyme to provide precursors of polyketide synthesis. The main research results are as follows:(1) Calow_0635 which encoded acyl CoA synthetase was found in the genome of C. Owensensis OL through sequence alignment, conserved motif analysis and phylogenetic tree construction. This gene was cloned into expression vector and heterologous expressed in Escherichia coli and purified through Ni affinity chromatograph.(2) It was proved that CoACS can recognize acetic acid, propionic acid, butyric acid, 2-methylpropionic acid, pentanoic acid, 2-methylbutyric acid, 3-methylbutyric acid and cyclohexanecarboxylic acid as substrates while cannot catalyze acetic acid, malonate acid and 2,3-dimethylacrylic acid. Through comparing the catalytic activity towards different carboxylic acids, it was found that CoACS has higher catalytic activity to pentanoic acid and butanoic acid than propionic acid, 2-methylpropionic acid, 2-methylbutanoicacid, and 3-methylbutanoic acid. The optimum reaction conditions were determined as 30 ℃, pH 7.0 using pentanoic acid and N-acetylcysteamine as substrates. It was also proved that CoACS remained 45% activity after incubation in 70 ℃ for eight hours. The kcat/Km value of pentanoic was 783.56 M-1s-1 which was much higher than other reported acyl CoA synthetases. (3) Through analyzing protein structures and amino acid sequences of several malonyl CoA synthetases, the reason why CoACS cannot recognize malonate acid was predicted as lacking three key amino acids which were conserved in all malonyl CoA synthetases. And furthermore, we proved the assumption through constructing a mutant of CoACS containing these three amino acids (F243H, A314S, and A336R). Activity test showed that this mutant can recognize malonate acid rather than pentanoic acid. The molecule of 2,3-dimethylacryl acid was docked into structure of benzoyl CoA synthetase (pdb:4EAT) to find out the reason that why CoACS cannot catalyze 2,3-dimethylacryl acid. Several residues which play key roles in stabilizing 2,3-dimethylacryl acid were found in benzoyl CoA synthetase. 

Document Type学位论文
Recommended Citation
GB/T 7714
董爽爽. 热稳定酰基辅酶A合成酶的异源表达和酶学特性研究[D]. 北京. 中国科学院研究生院,2016.
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