Structure of the fungal hydroxylase, CYP505A30, and rational transfer of mutation data from CYP102A1 to alter regioselectivity
Structure of the fungal hydroxylase, CYP505A30, and rational transfer of mutation data from CYP102A1 to alter regioselectivity
复制标题
真菌羟化酶 CYP505A30 的结构以及 CYP102A1 突变数据的合理转移以改变区域选择性
DOI：
10.1039/d1cy01348c
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发表时间：
2021
影响因子：
5
通讯作者：
Aschenbrenner J
中科院分区：
文献类型：
--
作者：
Aschenbrenner J
CYP 505 A30是一种真菌的、自给自足的细胞色素P450单加氧酶，可以选择性地氧化正烷烃、脂肪醇和脂肪酸。从烷烃，它通过两个连续的羟基化反应产生非邻位二醇的混合物。在这里，我们报告的血红素结构域的CYP 505 A30，第一个结构的CYP 505家族的成员，与十二烷酸结合在活性位点。总体而言，观察到与相关细菌CYP 102 A1的高结构相似性，尽管序列同一性低（<40%）。然而，活性位点的比较显示出高度的保守性，只有两个氨基酸的差异接近血红素。与CYP 102 A1一样，CYP 505 A30中的脂肪酸底物也通过精氨酸残基稳定。然而，与位于CYP 102 A1的β1区的R47相比，R358位于CYP 505 A30的β3区。我们还创建了突变体，以测试是否有可能合理地转移CYP 102 A1中活性位点突变的知识，以改变CYP 505 A30的区域选择性。F93 V、I334 F突变的引入导致ω-1（C2）区域选择性增加，与CYP 102 A1 87-328相似，对于正辛烷超过80%，对于正癸烷超过90%。改变残基以类似于CYP 102 A1野生型，使两种底物对ω-2（C3）的区域选择性增加至60%以上。从这项研究中获得的知识解锁一个更有选择性的生产对称的非邻位二醇从正构烷烃。
CYP505A30 is a fungal, self-sufficient cytochrome P450 monooxygenase that can selectively oxyfunctionalise n-alkanes, fatty alcohols, and fatty acids. From alkanes, it produces a mixture of non-vicinal diols by two sequential hydroxylation reactions. Here we report the structure of the haem domain of CYP505A30, the first structure for a member of the CYP505 family, with dodecanoic acid bound within the active site. Overall, a high structural similarity to the related bacterial CYP102A1 was observed, despite low sequence identity (<40%). Comparison of the active sites, however, showed a high degree of conservation with only two amino acid differences close to the haem. Stabilisation of the fatty acid substrate in CYP505A30 also occurs, as in CYP102A1, via an arginine residue. However, compared to R47, which is situated in the β1 region of CYP102A1, R358 is located in the β3 region of CYP505A30. We furthermore created mutants to test if it is possible to rationally transfer the knowledge on active site mutations in CYP102A1 to change the regioselectivity of CYP505A30. The introduction of F93V, I334F mutations resulted in increased ω-1 (C2) regioselectivity, similar to CYP102A1 87-328, of more than 80% for n-octane and 90% for n-decane. Changing residues to resemble the CYP102A1 wildtype increased the regioselectivity towards ω-2 (C3) to over 60% for both substrates. The knowledge gained from this study unlocks a more selective production of symmetrical non-vicinal diols from n-alkanes.