Amycolatosis CGMCC No1149 P450酶对洛伐他汀羟基化的影响及P450酶基因部分克隆
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摘要
他汀类药物属于羟甲基戊二酰辅酶A(HMG-CoA)还原酶抑制剂,为降脂药物中的首选药物,也是国内外研究的热点之一。无锡他汀(本实验室拥有自主知识产权)是一种新型HMG-CoA还原酶抑制剂,由洛伐他汀经拟无枝酸菌(Amycolatopsis sp.ST2710)转化产生。本文主要对Amycolatopsis sp.ST2710转化洛伐他汀为无锡他汀的转化过程中的相关酶进行了初步的研究,分析了转化过程中相关酶的酶活与洛伐他汀转化之间的关系,并克隆了该酶的部分基因序列,初步确定该酶为CYP450羟基化酶。
首先,在国内外文献的基础上,确立Amycolatopsis CGMCC No1149 P450酶测定方法为一氧化碳差光谱法,这一方法是根据还原型P450酶与CO结合以后在450nm处有一特征吸收峰的特性建立的。对Amycolatosis CGMCC No1149 P450酶测定方法进行了优化。主要考察了破壁方法,温度,pH以及缓冲体系对酶活测定的影响。最终确立酶活测定条件:以超声波破碎细胞获得细胞抽提液,温度为室温,缓冲体系为pH 7.4磷酸缓冲液,CO结合时间2 min,紫外分光光度计从400 nm向500 nm扫描。其中波长450 nm处的吸光率即代表了该CYPP450羟基化酶的酶活。
为了判断P450酶与底物羟基化的关系,考察了Amycolatopsis CGMCC No1149 P450酶对洛伐他汀转化率的影响。通过改变培养基初始pH,转化温度,底物浓度,溶氧等因素,考察P450酶活的变化及相应的洛伐他汀转化率的变化。结果发现,pH,温度,底物浓度,溶氧等均对P450的酶活有较大影响,其变化趋势与转化率的变化一致。同时,研究了在添加适量金属离子情况下P450酶活变化情况,其中添加Mg~(2+)、Fe~(2+)、Cu~(2+)后酶活有显著提高,同时对转化又明显的促进作用。研究结果表明,洛伐他汀的转化跟Amycolatopsis sp.ST2710中的细胞色素P450酶的酶活性密切相关。
通过对所查到的CYP450基因的序列进行分析,利用保守区进行简并PCR,克隆了P450酶基因部分基因片段,其长度为0.52 kb,经序列分析发现其与Amycolatopsis azurea CYP450基因核苷酸序列相似性最高,同源性达到了92%。对该基因片段所编码的氨基酸序列进行了分析,并与其他相关的CYP450氨基酸片段做了同源性比对,发现它们之间的同源性大都超过了60%,为同一亚家族成员。从而在分子生物学的角度初步判断CYP450是Amycolatopsis sp.ST2710转化洛伐他汀为无锡他汀过程中的一个相关酶。
Statins, which were inhibitors of 3-hydroxy-3-methylglutaryl coenzyme A(HMG-CoA) reductase, were the prime drugs of cardiovascular diseases. They were the focus of most research at both home and abroad. Wuxistatin, discovered by our research group, was a novel inhibitor of 3-hydroxy-3-methyl-glutaryl(HMG)-CoA reductase which was a rate-limiting enzyme of cholesterol biosynthesis. Lovastatin could be converted into wuxistatin by Amycolatopsis sp.ST2710. This paper mainly presents the enzymes involved in the process of bioconversion of lovastatin into wuxistatin by Amycolatopsis sp.ST2710 and analysis the relationship between the enzyme activity and the lovastatin bioconversion efficiency. And the genomic fragment of this enzyme was also cloned. All of these studies showed the key enzyme in the bioconversion of lovastatin by Amycolatopsis sp.ST2710 was a cytochrome P450 hydroxylase.
The method of enzymes activity assay was discussed in this study. According to the references, the CYP450 enzyme showed a typical absorption peak at 450nm after binded with CO, a method of reduced CO-difference spectrum was established to study the cytochrome P450 activity.
To obtain optimal assay condition, this method was optimized in our study. The methods of cell-wall broken, temperature, pH and buffer system were mainly investigated. And the optimized condition was: ultrasonic wave method, room temperature, pH 7.4, phosphate buffer, the CO-binding time was 2 min, ultraviolet spectrophotometer scanning from 400 nm to 500nm. The absorption peak at 450 nm expressed the enzyme activity of the CYP450 hydroxylase.
The relationship between CYP450 hydroxylase and the conversion efficiency was also studied by measuring the enzyme activity and the bioconversion of lovastatin in the converting process under different conditions. In the flask conditions, the initial pH of the medium, temperature, concentration of lovastain, shaking frequency, were the factors affecting the CYP450 enzyme activity, they showed the same effect to the bioconversion efficiency of lovastatin, respectively. The effects of metal ions to the CYP450 hydroxylase activity and the corresponding bioconversion were carried out. They also showed the same effect as they did to the bioconversion.
A 0.52 kb genomic fragment was cloned from Amycolatopsis sp.ST2710 using degenerate primers PCR, which homologous to CYP450 hydroxylase genes from other actinomycetes. Sequence analysis revealed that the 0.52 kb genomic fragment showed the highest identity to the CYP450 of Amycolatopsis azurea(92%).
首先,在国内外文献的基础上,确立Amycolatopsis CGMCC No1149 P450酶测定方法为一氧化碳差光谱法,这一方法是根据还原型P450酶与CO结合以后在450nm处有一特征吸收峰的特性建立的。对Amycolatosis CGMCC No1149 P450酶测定方法进行了优化。主要考察了破壁方法,温度,pH以及缓冲体系对酶活测定的影响。最终确立酶活测定条件:以超声波破碎细胞获得细胞抽提液,温度为室温,缓冲体系为pH 7.4磷酸缓冲液,CO结合时间2 min,紫外分光光度计从400 nm向500 nm扫描。其中波长450 nm处的吸光率即代表了该CYPP450羟基化酶的酶活。
为了判断P450酶与底物羟基化的关系,考察了Amycolatopsis CGMCC No1149 P450酶对洛伐他汀转化率的影响。通过改变培养基初始pH,转化温度,底物浓度,溶氧等因素,考察P450酶活的变化及相应的洛伐他汀转化率的变化。结果发现,pH,温度,底物浓度,溶氧等均对P450的酶活有较大影响,其变化趋势与转化率的变化一致。同时,研究了在添加适量金属离子情况下P450酶活变化情况,其中添加Mg~(2+)、Fe~(2+)、Cu~(2+)后酶活有显著提高,同时对转化又明显的促进作用。研究结果表明,洛伐他汀的转化跟Amycolatopsis sp.ST2710中的细胞色素P450酶的酶活性密切相关。
通过对所查到的CYP450基因的序列进行分析,利用保守区进行简并PCR,克隆了P450酶基因部分基因片段,其长度为0.52 kb,经序列分析发现其与Amycolatopsis azurea CYP450基因核苷酸序列相似性最高,同源性达到了92%。对该基因片段所编码的氨基酸序列进行了分析,并与其他相关的CYP450氨基酸片段做了同源性比对,发现它们之间的同源性大都超过了60%,为同一亚家族成员。从而在分子生物学的角度初步判断CYP450是Amycolatopsis sp.ST2710转化洛伐他汀为无锡他汀过程中的一个相关酶。
Statins, which were inhibitors of 3-hydroxy-3-methylglutaryl coenzyme A(HMG-CoA) reductase, were the prime drugs of cardiovascular diseases. They were the focus of most research at both home and abroad. Wuxistatin, discovered by our research group, was a novel inhibitor of 3-hydroxy-3-methyl-glutaryl(HMG)-CoA reductase which was a rate-limiting enzyme of cholesterol biosynthesis. Lovastatin could be converted into wuxistatin by Amycolatopsis sp.ST2710. This paper mainly presents the enzymes involved in the process of bioconversion of lovastatin into wuxistatin by Amycolatopsis sp.ST2710 and analysis the relationship between the enzyme activity and the lovastatin bioconversion efficiency. And the genomic fragment of this enzyme was also cloned. All of these studies showed the key enzyme in the bioconversion of lovastatin by Amycolatopsis sp.ST2710 was a cytochrome P450 hydroxylase.
The method of enzymes activity assay was discussed in this study. According to the references, the CYP450 enzyme showed a typical absorption peak at 450nm after binded with CO, a method of reduced CO-difference spectrum was established to study the cytochrome P450 activity.
To obtain optimal assay condition, this method was optimized in our study. The methods of cell-wall broken, temperature, pH and buffer system were mainly investigated. And the optimized condition was: ultrasonic wave method, room temperature, pH 7.4, phosphate buffer, the CO-binding time was 2 min, ultraviolet spectrophotometer scanning from 400 nm to 500nm. The absorption peak at 450 nm expressed the enzyme activity of the CYP450 hydroxylase.
The relationship between CYP450 hydroxylase and the conversion efficiency was also studied by measuring the enzyme activity and the bioconversion of lovastatin in the converting process under different conditions. In the flask conditions, the initial pH of the medium, temperature, concentration of lovastain, shaking frequency, were the factors affecting the CYP450 enzyme activity, they showed the same effect to the bioconversion efficiency of lovastatin, respectively. The effects of metal ions to the CYP450 hydroxylase activity and the corresponding bioconversion were carried out. They also showed the same effect as they did to the bioconversion.
A 0.52 kb genomic fragment was cloned from Amycolatopsis sp.ST2710 using degenerate primers PCR, which homologous to CYP450 hydroxylase genes from other actinomycetes. Sequence analysis revealed that the 0.52 kb genomic fragment showed the highest identity to the CYP450 of Amycolatopsis azurea(92%).
引文
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2.沈同,王镜岩.生物化学[M].下册.第3版.北京:高等教育出版社, 2002. 284-291.
3. Manzoni M, Rollini M. Biosynthesis and biotechnological production of statins by filamentous fungi and application of these cholesterol-lowering drugs[J]. Appl Microbiol Biotechnol, 2002, 58: 555-564.
4. Endo A, Kuroda M, Tsujita Y. ML-236A, ML-236B and ML-236C, new inhibitors of cholesterogenesis produced by Penicillium citrinum[J]. J Antibiot(Tokyo), 1976, 29: 1346-1348.
5. Brown MS, Faust JR, Goldstein JL, et al. Induction of 3-hydroxy-3-methylglutaryl coenzyme A reductase activity in human fibroblasts incubated compactin(ML-236B), a competitive inhibitor of the reductase[J]. J Biochem(Tokyo), 1978, 253: 1121-1128.
6. Endo A, Monacolin K. A new hypocholesterolemic agent produced by Monascus sp[J]. J Antibiot(Tokyo), 1979, 32: 852-854.
7. Endo A. Compactin(ML-236B) and related compounds as potential cholesterol-lowering agents that inhibit HMG-CoA reductase[J]. J Ind Microbiol Biotechnol, 1985, 28: 401- 405.
8. Endo A, Hasumi K. Dihydromonacolin L and Monacolin X, new metabolites those inhibit cholesterol biosynthesis[J]. J Antibiot(Tokyo), 1985, 38: 321-327.
9. Hoffman WF, Alberts AW, Anderson PS, et al. 3-hydroxy-3-methylglutaryl-coenzyme A reductase inhibitors is the side chain ester derivatives of mevinolin[J]. J Med Chem, 1986, 29: 849-852.
10. Tsujita Y, Kuroda M, Shimada Y, et al. CS-514, a competitive inhibitor of 3-hydroxy-3 -methylglutaryl coenzyme A reductase: Tissue-selective inhibition of sterol synthesis and hypolipidemic effect on various animal species[J]. Biochim Biophys Acta, 1986, 877(1): 50-60.
11. Serizawa N, Serizawa S, Nakagawa K, et al. Microbial hydroxylation of ML-236B (compactin).Studies on microorganisms capable of 3 beta-hydroxylation of ML-236B [J]. J Antibiot(Tokyo), 1983, 36(7):887-891.
12. Angerbauer R, Fey P, Hubsch W, et al. Effect of the new HMG-CoA reductase inhibitor cerivastatin(Bay W 6228)on migration, proliferation and cholesterol synthesis in arterial mycocytes[C]. XI International Symposium on Drugs Affecting Lipid Metabolism,Florence, Italy. Abs 69. 1992.
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