Re-Programming and Optimization of a L-Proline cis-4-Hydroxylase for the cis-3-Halogenation of its Native Substrate
Re-Programming and Optimization of a L-Proline cis-4-Hydroxylase for the cis-3-Halogenation of its Native Substrate
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DOI：
10.1002/cctc.202100591
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发表时间：
2021-06-29
期刊：
影响因子：
4.5
通讯作者：
Buller, Rebecca
中科院分区：
文献类型：
--
作者：
Papadopoulou, Athena;Meierhofer, Jasmin;Buller, Rebecca
作用于独立底物的非血红素铁/α-酮戊二酸依赖性卤化酶催化失活的C（sp（3））-H键的区域和立体选择性卤化。然而，只有少数这些卤化酶的特点，酶自由基卤化的生物合成潜力仍然有限。在此，我们描述了改造的L-脯氨酸顺式-4-羟化酶从苜蓿中华根瘤菌到卤化酶的酶的活性位点引入一个单点突变（D108 G）。重编程的卤化酶显示出惊人的区域发散反应化学：虽然L-脯氨酸的卤化仅发生在C3-位置，保留的羟基化活性导致衍生化在C4-位置，对应于野生型酶的区域选择性。通过采用几轮定向进化，鉴定了优化的卤化酶变体，与亲本酶SmP 4 H（D108 G）相比，其用于L-脯氨酸氯化的表观k（cat）/K-m提高了98倍。这种新型卤化生物催化剂的开发和优化突出了合理利用非血红素Fe/α KG依赖性双加氧酶的化学多功能性用于C-H官能化的可能性。
Non-heme iron/alpha-ketoglutarate dependent halogenases acting on freestanding substrates catalyze the regio- and stereoselective halogenation of inactivated C(sp(3))-H bonds. Yet, with only a handful of these halogenases characterized, the biosynthetic potential of enzymatic radical halogenation remains limited. Herein, we describe the remodeling of L-proline cis-4-hydroxylase from Sinorhizobium meliloti into a halogenase by introduction of a single point mutation (D108G) into the enzyme's active site. The re-programmed halogenase displays a striking regio-divergent reaction chemistry: While halogenation of L-proline exclusively occurs at the C3-position, the retained hydroxylation activity leads to derivatization at the C4-position, corresponding to the regioselectivity of the wildtype enzyme. By employing several rounds of directed evolution, an optimized halogenase variant with 98-fold improved apparent k(cat)/K-m for chlorination of L-proline compared to the parental enzyme SmP4H (D108G) was identified. The development and optimization of this novel halogenation biocatalyst highlights the possibility to rationally harness the chemical versatility of non-heme Fe/alpha KG dependent dioxygenases for C-H functionalization.