12.18细胞色素P450酶系ppt课件
相关主题
- 1、下载文档前请自行甄别文档内容的完整性，平台不提供额外的编辑、内容补充、找答案等附加服务。
- 2、"仅部分预览"的文档,不可在线预览部分如存在完整性等问题,可反馈申请退款(可完整预览的文档不适用该条件!)。
- 3、如文档侵犯您的权益，请联系客服反馈,我们会尽快为您处理(人工客服工作时间：9:00-18:30)。
• No side product formation could be observed when resting cells were used as biocatalysts.
Comparison of growing and resting cells for A) progesterone and B) testosterone conversion
细胞色素P450酶系
孙婉菊 2014年12月17日
1.细胞色素P450酶系概述
• 细胞色素P450单加氧酶系(cytochrome P450 monooxygenases，P450s) ： NAD(P)H-细胞色素P450还原酶 (CPR) 细胞色素P450单加氧酶 (CYP) 2个组分均通过疏水的膜结合锚点结合在微粒体膜上。
• 甾体化合物分子中11-羟基的引入在甾体药物化学中占极为重要的地位 。
eg1
Towards Preparative Scale Steroid Hydroxylation with Cytochrome P450 Monooxygenase CYP106A2
1.CYP106A2 from Bacillus megaterium ATCC 13368, enables the oxidation of 3-keto-4-ene steroids mainly at position 15.
2.We expressed this enzyme together with the electron-transfer partners bovine adrenodoxin and adrenodoxin reductase in Escherichia coli.
3.An enzyme-coupled cofactor regeneration system was implemented by expressing alcohol dehydrogenase from Lactobacillus brevis。
3.甾体羟化反应
• 甾体羟化酶大都是细胞色素P450依赖型单加氧酶，属于末端氧 化酶，能将一摩尔分子氧引入底物，通过NADPH递氢，完成羟 化反应。
RH + NADPH + O2
ROH + NADP+ + OH-
Steroidogenesis
• 细胞色素P450酶系羟化反应主要有：9α羟化、11α羟化、11β羟化、 16α羟化、17α羟化和19α羟化等。
• CYP450 广泛分布在动物、植物、低等真核生物和细菌体内，在同种 生物的不同组织中都存在，是自然界中含量最丰富、底物谱最广的催 化酶系。
NADPH-细胞色素P450 还原酶
• NADPH-细胞色素P450还原酶是一种以FAD和FMN作为辅基的细胞微粒体 复合黄素蛋白； CYP450是以硫醇盐结合血红素为活性中心催化氧原 子转移的氧化还原酶，氧由亚铁血红素（HEME）来活化。
Substrate solubility and transport into the cell
53% 21%
• Highest activity was observed in presence of even 2m (15.4 vol%) propan-2-ol.
• During the second reaction
• CPR是CYP的主要电子供体，电子传递给CYP之后，CYP才能与底物发生 氧化还原反应，发挥代谢活性，CPR与CYPHale Waihona Puke Baidu电子传递反应是CYP氧化 还原反应的限速步骤 。
2.Human and Microbial CYPs
• Human CYPs Humans contain 57 CYPs mitochond rial CYPs: CYP11A1、CYP11B1、CYP11B2 microsomal CYPs: CYP17A1、CYP19A1、CYP21A1 Six P450 enzymes are involved in steroidogenesis
Cloning
• pBAR Twin: bovine AdR、Adx • pACYC FHH28: CYP106A2 • pBAR Tripel: bovine AdR、AdX、LbADH
Selectivity of CYP106A2
• With growing cells as a biocatalyst, a limited selectivity of both Prog and Tes hydroxylation was observed.
cycle only 21% of Prog could be converted in the presence of propan-2-ol. Without this cosolvent
53% 15b-Prog was formed;
• This indicates the limited
applicability of propan-2-ol as a cosolvent in this system.
• Microbial CYPs Actinomycetes Mycobacterium smegmatis (39 P450s) Mycobacterium tuberculosis (20 P450s) Streptomyces coelicolor (18 P450s)
In human adrenal P450s been intensively studied in steroid synthesis. In bacteria, the P450s are used in catabolic pathways for the production of steroids, bioactive lipids and chiral synthons.
Influence of propan-2-ol on the activity (A) and stability (B) of the wh ole-cell biocatalyst
Comparison of growing and resting cells for A) progesterone and B) testosterone conversion
细胞色素P450酶系
孙婉菊 2014年12月17日
1.细胞色素P450酶系概述
• 细胞色素P450单加氧酶系(cytochrome P450 monooxygenases，P450s) ： NAD(P)H-细胞色素P450还原酶 (CPR) 细胞色素P450单加氧酶 (CYP) 2个组分均通过疏水的膜结合锚点结合在微粒体膜上。
• 甾体化合物分子中11-羟基的引入在甾体药物化学中占极为重要的地位 。
eg1
Towards Preparative Scale Steroid Hydroxylation with Cytochrome P450 Monooxygenase CYP106A2
1.CYP106A2 from Bacillus megaterium ATCC 13368, enables the oxidation of 3-keto-4-ene steroids mainly at position 15.
2.We expressed this enzyme together with the electron-transfer partners bovine adrenodoxin and adrenodoxin reductase in Escherichia coli.
3.An enzyme-coupled cofactor regeneration system was implemented by expressing alcohol dehydrogenase from Lactobacillus brevis。
3.甾体羟化反应
• 甾体羟化酶大都是细胞色素P450依赖型单加氧酶，属于末端氧 化酶，能将一摩尔分子氧引入底物，通过NADPH递氢，完成羟 化反应。
RH + NADPH + O2
ROH + NADP+ + OH-
Steroidogenesis
• 细胞色素P450酶系羟化反应主要有：9α羟化、11α羟化、11β羟化、 16α羟化、17α羟化和19α羟化等。
• CYP450 广泛分布在动物、植物、低等真核生物和细菌体内，在同种 生物的不同组织中都存在，是自然界中含量最丰富、底物谱最广的催 化酶系。
NADPH-细胞色素P450 还原酶
• NADPH-细胞色素P450还原酶是一种以FAD和FMN作为辅基的细胞微粒体 复合黄素蛋白； CYP450是以硫醇盐结合血红素为活性中心催化氧原 子转移的氧化还原酶，氧由亚铁血红素（HEME）来活化。
Substrate solubility and transport into the cell
53% 21%
• Highest activity was observed in presence of even 2m (15.4 vol%) propan-2-ol.
• During the second reaction
• CPR是CYP的主要电子供体，电子传递给CYP之后，CYP才能与底物发生 氧化还原反应，发挥代谢活性，CPR与CYPHale Waihona Puke Baidu电子传递反应是CYP氧化 还原反应的限速步骤 。
2.Human and Microbial CYPs
• Human CYPs Humans contain 57 CYPs mitochond rial CYPs: CYP11A1、CYP11B1、CYP11B2 microsomal CYPs: CYP17A1、CYP19A1、CYP21A1 Six P450 enzymes are involved in steroidogenesis
Cloning
• pBAR Twin: bovine AdR、Adx • pACYC FHH28: CYP106A2 • pBAR Tripel: bovine AdR、AdX、LbADH
Selectivity of CYP106A2
• With growing cells as a biocatalyst, a limited selectivity of both Prog and Tes hydroxylation was observed.
cycle only 21% of Prog could be converted in the presence of propan-2-ol. Without this cosolvent
53% 15b-Prog was formed;
• This indicates the limited
applicability of propan-2-ol as a cosolvent in this system.
• Microbial CYPs Actinomycetes Mycobacterium smegmatis (39 P450s) Mycobacterium tuberculosis (20 P450s) Streptomyces coelicolor (18 P450s)
In human adrenal P450s been intensively studied in steroid synthesis. In bacteria, the P450s are used in catabolic pathways for the production of steroids, bioactive lipids and chiral synthons.
Influence of propan-2-ol on the activity (A) and stability (B) of the wh ole-cell biocatalyst