乳糖诱导剂促进P450 BM-3在大肠杆菌中可溶性表达的研究

P450 BM-3是一种具有工业化应用潜力的单加氧酶,可催化饱和脂肪酸羟基化。为提高其在大肠杆菌宿主中的可溶性表达水平,采用乳糖作为诱导剂对P450 BM-3的诱导表达条件进行研究。结果发现：在大肠杆菌的OD600达到0.7~1.5时,添加2.0 g/L的乳糖、30℃诱导10 h可获得最佳诱导效果。与IP TG的诱导效果对比发现：采用乳糖作诱导剂时,菌体生物量提高1.09倍,目标蛋白量提升2.13倍,蛋白包涵体的比例则降低至10%。研究结果表明：乳糖可显著提升P450 BM-3在大肠杆菌中的重组表达水平,并且能够促进p450 BM-3的可溶性表达。     

生物酶法催化瓦伦西亚烯生成圆柚酮

在体外,利用野生型CYP450BM-3对瓦伦西亚烯进行催化,酶-底物复合物催化NADPH氧化的速率为31±1.0 nmol(nmol P450)-1min-1,但催化产物中没有检测到圆柚酮的生成。突变体R47L/Y51F/F87A与底物复合物催化NADPH氧化的速率高于野生型,为79±6.5 nmol(nmol P450)-1min-1,并在催化产物中检测到圆柚酮的生成,但其产物选择性较差,圆柚酮的含量仅占总产物的6.8%。与此同时,检测了另一个突变体A74G/F87V/L188Q对瓦伦西亚烯的催化效果,发现其与底物复合物对NADPH的氧化速率与突变体R47L/Y51F/F87A相当,但产物中圆柚酮的比率更高,达8.0%。     

过氧化氢驱动的细胞色素P450酶研究进展

细胞色素P450酶在自然界中广泛存在,能催化多种类型的氧化反应,在有机合成和生物化工方面具有重要的应用潜力。尽管大多数P450酶通常需要辅酶和复杂的电子传递体系协助活化氧分子,一些P450酶也可以利用过氧化氢作为末端氧化剂,这极大地简化了催化循环,为P450酶的合成应用提供了一条新的简便途径。本文系统地介绍了几类过氧化氢驱动的P450酶催化体系,包括脂肪酸羟化酶P450SPα和P450BSβ、脂肪酸脱羧酶P450OleTJE、人工改造的羟化酶P450BM3和P450cam突变体、以及基于底物误识别策略的P450-H2O2体系。通过分析催化反应机制,本文探讨了P450-H2O2催化体系在目前存在的挑战和可能的解决途径,并对其进一步应用前景进行了展望。     

过氧化氢驱动的细胞色素P450酶研究进展

细胞色素P450酶在自然界中广泛存在,能催化多种类型的氧化反应,在有机合成和生物化工方面具有重要的应用潜力。尽管大多数P450酶通常需要辅酶和复杂的电子传递体系协助活化氧分子,一些P450酶也可以利用过氧化氢作为末端氧化剂,这极大地简化了催化循环,为P450酶的合成应用提供了一条新的简便途径。本文系统地介绍了几类过氧化氢驱动的P450酶催化体系,包括脂肪酸羟化酶P450SPα和P450BSβ、脂肪酸脱羧酶P450OleTJE、人工改造的羟化酶P450BM3和P450cam突变体、以及基于底物误识别策略的P450-H2O2体系。通过分析催化反应机制,本文探讨了P450-H2O2催化体系在目前存在的挑战和可能的解决途径,并对其进一步应用前景进行了展望。     

催化吲哚生成靛蓝的细胞色素P450BM-3 定向进化研究

以催化吲哚产生的靛蓝在 630 nm 处具有特殊的吸收峰为高通量筛选指标,将来源于 Bacillus megaterium 的细胞色素 P450BM-3 单加氧酶的基因序列用易错聚合酶链式反应进行定向进化,通过多轮突变,在原有的能产靛蓝的高活力突变酶的基础上成功获得了三个高于亲本酶的突变酶,突变酶的酶活分别是亲本酶的 6.6 倍 (hml001) , 9.6 倍 (hml002) 和 5.3 倍 (hml003) ,并对突变酶的动力学参数进行了分析 . 突变酶 DNA 测序的结果表明, hml001 含有一个有义氨基酸置换 I39V , hml002 含有三个有义氨基酸置换 D168N , A225V , K440N , hml003 含有一个有义氨基酸置换 E435D ,这些突变位点有些远离底物结合部位,有些位于底物结合部位 .     

P450cam突变体蛋白表达、纯化与结晶的改进及其四突变体（F87L/Y96F/L244A/V247A）晶体结构

在P450cam突变体蛋白纯化过程中使用280 nm/392 nm双波长比值检测蛋白质纯度, 采用β-巯基乙醇对蛋白质进行还原性保护,有效地缩短了纯化进程,纯化效率相应提高.以PEG 8000为沉淀剂经悬滴汽相扩散法筛选得到适合衍射的P450cam四突变体（F87L/Y96F/L244A/V247A）晶体,在Mar-Research面探测器系统上收集了0.22 nm分辨率的X射线衍射数据.采用同晶差值傅立叶法解析结构,最后的晶体学R因子和R_free分别为0.197和0.247,键长偏差为0.001 77 nm,键角偏差为1.96°.结构测定显示P450cam四突变体（F87L/Y96F/L244A/V247A）和P450cam野生型的整体构象无重大变化,突变后活性口袋变大而疏水性增加,这与突变设计预期目标一致.     

理性设计提高酯合成催化反应脂肪酶的热稳定性

【背景】南极假丝酵母脂肪酶B (Candida antarctica lipase B,CALB)具有优异的酯合成活性,是在非水相催化中应用极为广泛的工业用酶。【目的】在保留CALB优秀催化性能的基础上,提高CALB的热稳定性。【方法】采用预测软件PoPMuSiC和FoldX计算CALB潜在热稳定性突变位点,并根据氨基酸残基的空间位置进一步筛选。利用重叠延伸PCR技术在基因calb中引入10个单点突变,于毕赤酵母GS115中表达。【结果】点突变A146G、A151P、L278M均能有效提高CALB的热稳定性。在单点突变的基础上,组合突变体A146G-L278M和A146G-L278M-A151P的热稳定性得到进一步提高。与野生型相比,突变体A146G-L278M和A146G-L278M-A151P的最适反应温度均提高了5°C,T_m值分别提高了3.3°C和4.2°C。此外,合成己酸乙酯的酶促反应动力学分析表明,相比于野生型,突变体A146G-L278M和A146G-L278M-A151P对己酸和乙醇均具有更高的亲和力,且对己酸的催化效率k_(catA)/K_(m A)是野生型的4.1倍。通过分子动力学模拟,从分子水平阐明了突变体A146G-L278M和A146G-L278M-A151P热稳定性提高的机制。【结论】本研究采用的理性设计策略对提高CALB的热稳定性是行之有效的,该策略可作为其他工业用酶提高热稳定性的参考。     

共表达极端菌伴侣蛋白prefoldin提高P450 BM3突变体催化效率

P450 BM3来源于巨大芽孢杆菌,其突变体(A74G、F87V、L188Q、D168H)能够在大肠杆菌细胞内催化吲哚合成靛蓝．然而在常规的培养条件下(37℃,250 r/min),大肠杆菌细胞内靛蓝的生物转化量极低．本文将极端嗜热古菌Pyrococcus furiosus的分子伴侣蛋白与P450 BM3突变体在大肠杆菌进行共表达,以研究分子伴侣蛋白是否能够提高靛蓝的生物转化量．实验结果表明,极端嗜热古菌的分子伴侣蛋白prefoldin能够显著提高靛蓝的产量．同时,实验结果发现prefoldin能够明显提高大肠杆菌细胞内的NADPH/NADP+比率．鉴于NADPH是参与靛蓝生物转化过程的重要因素,靛蓝生物转化量的显著增加可能与该比率的提高有关．     

共表达极端菌伴侣蛋白prefoldin提高P450 BM3突变体催化效率（英文）

P450 BM3来源于巨大芽孢杆菌,其突变体(A74G、F87V、L188Q、D168H)能够在大肠杆菌细胞内催化吲哚合成靛蓝.然而在常规的培养条件下(37℃,250 r/min),大肠杆菌细胞内靛蓝的生物转化量极低.本文将极端嗜热古菌Pyrococcus furiosus的分子伴侣蛋白与P450 BM3突变体在大肠杆菌进行共表达,以研究分子伴侣蛋白是否能够提高靛蓝的生物转化量.实验结果表明,极端嗜热古菌的分子伴侣蛋白prefoldin能够显著提高靛蓝的产量.同时,实验结果发现prefoldin能够明显提高大肠杆菌细胞内的NADPH/NADP~+比率.鉴于NADPH是参与靛蓝生物转化过程的重要因素,靛蓝生物转化量的显著增加可能与该比率的提高有关.     

D-泛解酸内酯水解酶的定向进化

易错PCR结合DNA改组方法向D-泛解酸内酯水解酶基因中引入突变,并构建突变体库。利用酶的催化特点和产物特性建立了基于平板初筛和高效液相复筛的两步法D-泛解酸内酯水解酶活性筛选系统。用该筛选系统以酶活力和pH稳定性为指标对突变体库进行筛选,最终获得一株酶活力高且在低pH条件下稳定性好的突变体Mut E-861。该突变体的酶活力是野生型酶的5.5倍。对突变体和野生型酶在pH 6.0和pH 5.0条件下的残余酶活进行对比,在这两种pH条件下,突变体酶的酶活残留分别为75%和50%,而野生型酶只能保持原来的40%和20%。通过软件对突变体Mut E-861酶基因和野生型酶基因进行分析对比,发现突变体Mut E-861酶基因发生了三处点突变,其中突变使两处氨基酸取代,另一处为沉默突变,未引起氨基酸的变化。     

Altering the regioselectivity of the subterminal fatty acid hydroxylase P450 BM-3 towards gamma- and delta-positions

Cytochrome P450 BM-3 monooxygenase from Bacillus megaterium (CYP102A1) catalyzes the subterminal hydroxylation of fatty acids with a chain length of 12-22 carbons. Wild-type P450 BM-3 oxidizes saturated fatty acids at subterminal positions producing a mixture of omega-1, omega-2 and omega-3 hydroxylated products. Using a rational site-directed mutagenesis approach, three new elements have been introduced into the substrate binding pocket of the monooxygenase, which greatly changed the product pattern of lauric acid hydroxylation. Particularly, substitutions at positions S72, V78 and I263 had an effect on the enzyme regioselectivity. The P450 BM-3 mutants V78A F87A I263G and S72Y V78A F87A were able to oxidize lauric acid not only at delta-position (14% and 16%, respectively), but also produced gamma- and beta-hydroxylated products. delta-Hydroxy lauric and gamma-hydroxy lauric acid are important synthons for the production of the corresponding lactones.     

Rational evolution of a medium chain-specific cytochrome P-450 BM-3 variant

The single mutant F87A of cytochrome P-450 BM-3 from Bacillus megaterium was engineered by rational evolution to achieve improved hydroxylation activity for medium chain length substrates (C8-C10). Rational evolution combines rational design and directed evolution to overcome the drawbacks of these methods when applied individually. Based on the X-ray structure of the enzyme, eight mutation sites (P25, V26, R47, Y51, S72, A74, L188, and M354) were identified by modeling. Sublibraries created by site-specific randomization mutagenesis of each single site were screened using a spectroscopic assay based on omega-p-nitrophenoxycarboxylic acids (pNCA). The mutants showing activity for shorter chain length substrates were combined, and these combi-libraries were screened again for mutants with even better catalytic properties. Using this approach, a P-450 BM-3 variant with five mutations (V26T, R47F, A74G, L188K, and F87A) that efficiently hydrolyzes 8-pNCA was obtained. The catalytic efficiency of this mutant towards omega-p-nitrophenoxydecanoic acid (10-pNCA) and omega-p-nitrophenoxydodecanoic acid (12-pNCA) is comparable to that of the wild-type P-450 BM-3.     

Modelling of three-dimensional structures of cytochromes P450 11B1 and 11B2.

The final steps of the biosynthesis of glucocorticoids and mineralocorticoids in the adrenal cortex require the action of two different cytochromes P450--CYP11B1 and CYP11B2. Homology modelling of the three-dimensional structures of these cytochromes was performed based on crystallographic coordinates of two bacterial P450s, CYP102 (P450BM-3) and CYP108 (P450terp). Principal attention was given to the modelling of the active sites and a comparison of the active site structures of CYP11B1 and CYP11B2 was performed. It can be demonstrated that key residue contacts within the active site appear to depend on the orientation of the heme. The obtained 3D structures of CYP11B1 and CYP11B2 were used for investigation of structure-function relationships of these enzymes. Previously obtained results on naturally occurring mutants and on mutants obtained by site-directed mutagenesis are discussed.     

Sensitive assay for laboratory evolution of hydroxylases toward aromatic and heterocyclic compounds

Powerful directed evolution methods have been developed for tailoring proteins to our needs in industrial applications. Here, the authors report a medium-throughput assay system designed for screening mutant libraries of oxygenases capable of inserting a hydroxyl group into a C-H bond of aromatic or O-heterocyclic compounds and for exploring the substrate profile of oxygenases. The assay system is based on 4-aminoantipyrine (4-AAP), a colorimetric phenol detection reagent. By using 2 detection wavelengths (509 nm and 600 nm), the authors achieved a linear response from 50 to 800 microM phenol and standard deviations below 11% in 96-well plate assays. The monooxygenase P450 BM-3 and its F87A mutant were used as a model system for medium-throughput assay development, identification of novel substrates (e.g., phenoxytoluene, phenylallyether, and coumarone), and discovery of P450 BM-3 F87A mutants with 8-fold improvement in 3-phenoxytoluene hydroxylation activity. This activity increase was achieved by screening a saturation mutagenesis library of amino acid position Y51 using the 4-AAP protocol in the 96-well format.     

Directed evolution of cytochrome P450 for sterol epoxidation

16,17-Epoxysterol plays an important role in pharmaceutical steroid synthesis. To investigate the potential application of cytochrome P450 for epoxysterol synthesis, an approach to the epoxidation of 16,17-epoxysterol, based on directed evolution of cytochrome P450 BM-3, was developed. This comprised random gene mutagenesis for optimizing the activity of P450 BM-3 for epoxidation of hydrophobic sterol, followed by the 7-ethoxycoumarin de-ethylation assay for general enzyme activity detection and the modified picric acid assay for epoxidation activity screening. By the two-step screening, one mutant from 792 clones showed specific substrate activity of converting progesterone to 16,17-epoxysterol, which validated the possibility to evolve the cytochrome P450 for the synthesis of steroidal epoxides.     

A single active-site mutation of P450BM-3 dramatically enhances substrate binding and rate of product formation

Identifying key structural features of cytochromes P450 is critical in understanding the catalytic mechanism of these important drug-metabolizing enzymes. Cytochrome P450BM-3 (BM-3), a structural and mechanistic P450 model, catalyzes the regio- and stereoselective hydroxylation of fatty acids. Recent work has demonstrated the importance of water in the mechanism of BM-3, and site-specific mutagenesis has helped to elucidate mechanisms of substrate recognition, binding, and product formation. One of the amino acids identified as playing a key role in the active site of BM-3 is alanine 328, which is located in the loop between the K helix and β 1-4. In the A328V BM-3 mutant, substrate affinity increases 5-10-fold and the turnover number increases 2-8-fold compared to wild-type enzyme. Unlike wild-type enzyme, this mutant is purified from E. coli with endogenous substrate bound due to the higher binding affinity. Close examination of the crystal structures of the substrate-bound native and A328V mutant BMPs indicates that the positioning of the substrate is essentially identical in the two forms of the enzyme, with the two valine methyl groups occupying voids present in the active site of the wild-type substrate-bound structure.     

Cytochrome P450BM-3 reduces aldehydes to alcohols through a direct hydride transfer

Cytochrome P450BM-3 catalyzed the reduction of lipophilic aldehydes to alcohols efficiently. A k(cat) of ～25 min(-1) was obtained for the reduction of methoxy benzaldehyde with wild type P450BM-3 protein which was higher than in the isolated reductase domain (BMR) alone and increased in specific P450-domain variants. The reduction was caused by a direct hydride transfer from preferentially R-NADP(2)H to the carbonyl moiety of the substrate. Weak substrate-P450-binding of the aldehyde, turnover with the reductase domain alone, a deuterium incorporation in the product from NADP(2)H but not D(2)O, and no inhibition by imidazole suggests the reductase domain of P450BM-3 as the potential catalytic site. However, increased aldehyde reduction by P450 domain variants (P450BM-3 F87A T268A) may involve allosteric or redox mechanistic interactions between heme and reductase domains. This is a novel reduction of aldehydes by P450BM-3 involving a direct hydride transfer and could have implications for the metabolism of endogenous substrates or xenobiotics.     

A P450 BM-3 mutant hydroxylates alkanes, cycloalkanes, arenes and heteroarenes

P450 monooxygenases from microorganisms, similar to those of eukaryotic mitochondria, display a rather narrow substrate specificity. For native P450 BM-3, no other substrates than fatty acids or an indolyl-fatty acid derivative have been reported (Li, Q.S., Schwaneberg, U., Fischer, P., Schmid, R.D., 2000. Directed evolution of the fatty-acid hydroxylase P450BM-3 into an indole-hydroxylating catalyst. Chem. Eur. J. 6 (9), 1531-1536). Engineering the substrate specificity of Bacillus megaterium cytochrome P-450 BM3: hydroxylation of alkyl trimethylammonium compounds. Biochem. J. 327, 537-544). We thus were quite surprised to observe, in the course of our investigations on the rational evolution of this enzyme towards mutants, capable of hydroxylating shorter-chain fatty acids, that a triple mutant P450 BM-3 (Phe87Val, Leu188-Gln, Ala74Gly, BM-3 mutant) could efficiently hydroxylate indole, leading to the formation of indigo and indirubin (Li, Q.S., Schwaneberg, U., Fischer, P., Schmid, R.D., 2000. Directed evolution of the fatty-acid hydroxylase P450BM-3 into an indole-hydroxylating catalyst. Chem. Eur. J. 6 (9), 1531-1536). Indole is not oxidized by the wild-type enzyme; it lacks the carboxylate group by which the proper fatty acid substrates are supposed to be bound at the active site of the native enzyme, via hydrogen bonds to the charged amino acid residues Arg47 and Tyr51. Our attempts to predict the putative binding mode of indole to P450 BM-3 or the triple mutant by molecular dynamics simulations did not provide any useful clue. Encouraged by the unexpected activity of the triple mutant towards indole, we investigated in a preliminary, but systematic manner several alkanes, alicyclic, aromatic, and heterocyclic compounds, all of which are unaffected by the native enzyme, for their potential as substrates. We here report that this triple mutant indeed is capable to hydroxylate a respectable range of other substrates, all of which bear little or no resemblance to the fatty acid substrates of the native enzyme.     

Interactions of substrates at the surface of P450s can greatly enhance substrate potency

Cytochrome P450s are a superfamily of heme containing enzymes that use molecular oxygen and electrons from reduced nicotinamide cofactors to monooxygenate organic substrates. The fatty acid hydroxylase P450BM-3 has been particularly widely studied due to its stability, high activity, similarity to mammalian P450s, and presence of a cytochrome P450 reductase domain that allows the enzyme to directly receive electrons from NADPH without a requirement for additional redox proteins. We previously characterized the substrate N-palmitoylglycine, which found extensive use in studies of P450BM-3 due to its high affinity, high turnover number, and increased solubility as compared to fatty acid substrates. Here, we report that even higher affinity substrates can be designed by acylation of other amino acids, resulting in P450BM-3 substrates with dissociation constants below 100 nM. N-Palmitoyl-l-leucine and N-palmitoyl-l-methionine were found to have the highest affinity, with dissociation constants of less than 8 nM and turnover numbers similar to palmitic acid and N-palmitoylglycine. The interactions of the amino acid side chains with a hydrophobic pocket near R47, as revealed by our crystal structure determination of N-palmitoyl-l-methionine bound to the heme domain of P450BM-3, appears to be responsible for increasing the affinity of substrates. The side chain of R47, previously shown to be important in interactions with negatively charged substrates, does not interact strongly with N-palmitoyl-l-methionine and is found positioned at the enzyme-solvent interface. These are the tightest binding substrates for P450BM-3 reported to date, and the affinity likely approaches the maximum attainable affinity for the binding of substrates of this size to P450BM-3.