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Directed OmniChange Evolution Converts P450 BM3 into an Alkyltrimethylammonium Hydroxylase
Chemistry - A European Journal ( IF 3.9 ) Pub Date : 2018-10-17 , DOI: 10.1002/chem.201803806 Yu Ji 1 , Alan Maurice Mertens 1 , Christoph Gertler 1 , Sallama Fekiri 1 , Merve Keser 1 , Daniel F. Sauer 1 , Kilian E. C. Smith 2 , Ulrich Schwaneberg 1, 3
Chemistry - A European Journal ( IF 3.9 ) Pub Date : 2018-10-17 , DOI: 10.1002/chem.201803806 Yu Ji 1 , Alan Maurice Mertens 1 , Christoph Gertler 1 , Sallama Fekiri 1 , Merve Keser 1 , Daniel F. Sauer 1 , Kilian E. C. Smith 2 , Ulrich Schwaneberg 1, 3
Affiliation
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Cetyl‐trimethylammonium bromide (CTAB) is a widely used cationic surfactant that is biodegradable in nature. CTAB biodegradation requires hydroxylation in the first step, which is rate‐limiting and crucial for solubility in water. In this study, the OmniChange multi‐site mutagenesis method was applied to reengineer the P450 BM3 substrate specificity towards the hydroxylation of CTAB by simultaneous mutagenesis of four previously reported positions (R47, Y51, F87, and L188). 1740 clones from the P450 BM3 OmniChange library were screened with the NADPH depletion assay. A total of 696 clones were rescreened with the NADPH depletion and an Ampliflu™ Red/ horseradish peroxidase based H2O2 detection assay. Several improved P450 BM3 variants were identified and finally four were kinetically characterized with respect to CTAB hydroxylation, based on both performance and coupling efficiency. Based on NADPH consumption, the P450 BM3 variant P3A8 (R47E/Y51M/F87V/L188E) displayed an initial activity (64.9±4.8 s−1, 13.5‐fold increased activity compared with wild‐type P450 BM3), which nearly matches the specific activity for its natural fatty acid substrate (palmitic acid (32–122 s−1)). Variant P3A8 showed high coupling efficiency (92.5 %), whereas wild‐type P450 BM3 displayed a low coupling efficiency (0.5 %). HPLC‐MS/MS detection confirmed that P3A8 and P2E7 (R47D/Y51L/F87V/L188A) form 13 and 35 times more 2‐hydroxylated CTAB than P450 BM3. In addition, di‐hydroxylated CTAB products were detected for all four investigated P450 BM3 variants (up to a yield of 77 %; P3A8). Di‐hydroxylated quaternary amines are highly interesting bolaform surfactants with a high hydrophilicity (surface contact angle: θ=16.7°).
中文翻译：
指导OmniChange Evolution将P450 BM3转化为烷基三甲基铵羟化酶
十六烷基三甲基溴化铵（CTAB）是一种广泛使用的阳离子表面活性剂，可自然降解。CTAB生物降解的第一步需要进行羟基化，这是限速的，对于在水中的溶解度至关重要。在这项研究中，通过同时诱变四个先前报道的位点（R47，Y51，F87和L188），使用OmniChange多位点诱变方法重新构建了针对CTAB羟基化的P450 BM3底物特异性。使用NADPH耗竭试验筛选了来自P450 BM3 OmniChange库的1740个克隆。使用NADPH耗竭和基于Ampliflu™Red /辣根过氧化物酶的H 2 O 2重新筛选总共696个克隆检测分析。确定了几个改进的P450 BM3变体，最后根据性能和偶联效率对CTAB羟基化进行了动力学表征。根据NADPH消耗量，P450 BM3变体P3A8（R47E / Y51M / F87V / L188E）表现出初始活性（与野生型P450 BM3相比，活性增加了64.9±4.8 s -1，活性增加了13.5倍），几乎与天然脂肪酸底物（棕榈酸（32–122 s -1））。变体P3A8显示出高耦合效率（92.5％），而野生型P450 BM3显示出低耦合效率（0.5％）。HPLC-MS / MS检测证实，P3A8和P2E7（R47D / Y51L / F87V / L188A）形成的2-羟基化CTAB数量是P450 BM3的13倍和35倍。此外，对于所有四个研究的P450 BM3变体都检测到二羟基化CTAB产物（产率高达77％； P3A8）。二羟基化的季胺是高度有趣的具有高亲水性（表面接触角：θ = 16.7°）的硼酸盐表面活性剂。
更新日期：2018-10-17
中文翻译：
指导OmniChange Evolution将P450 BM3转化为烷基三甲基铵羟化酶
十六烷基三甲基溴化铵（CTAB）是一种广泛使用的阳离子表面活性剂，可自然降解。CTAB生物降解的第一步需要进行羟基化，这是限速的，对于在水中的溶解度至关重要。在这项研究中，通过同时诱变四个先前报道的位点（R47，Y51，F87和L188），使用OmniChange多位点诱变方法重新构建了针对CTAB羟基化的P450 BM3底物特异性。使用NADPH耗竭试验筛选了来自P450 BM3 OmniChange库的1740个克隆。使用NADPH耗竭和基于Ampliflu™Red /辣根过氧化物酶的H 2 O 2重新筛选总共696个克隆检测分析。确定了几个改进的P450 BM3变体，最后根据性能和偶联效率对CTAB羟基化进行了动力学表征。根据NADPH消耗量，P450 BM3变体P3A8（R47E / Y51M / F87V / L188E）表现出初始活性（与野生型P450 BM3相比，活性增加了64.9±4.8 s -1，活性增加了13.5倍），几乎与天然脂肪酸底物（棕榈酸（32–122 s -1））。变体P3A8显示出高耦合效率（92.5％），而野生型P450 BM3显示出低耦合效率（0.5％）。HPLC-MS / MS检测证实，P3A8和P2E7（R47D / Y51L / F87V / L188A）形成的2-羟基化CTAB数量是P450 BM3的13倍和35倍。此外，对于所有四个研究的P450 BM3变体都检测到二羟基化CTAB产物（产率高达77％； P3A8）。二羟基化的季胺是高度有趣的具有高亲水性（表面接触角：θ = 16.7°）的硼酸盐表面活性剂。
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