Bloomer Brandon J, Natoli Sean N, Garcia-Borràs Marc, Pereira Jose H, Hu Derek B, Adams Paul D, Houk K N, Clark Douglas S, Hartwig John F
Department of Chemistry, University of California, Berkeley, CA, USA.
Chemical Sciences Division, Lawrence Berkeley National Laboratory, Berkeley, CA, USA.
Nat Catal. 2023 Jan;6(1):39-51. doi: 10.1038/s41929-022-00899-9. Epub 2023 Jan 12.
Artificial metalloenzymes (ArMs), which contain non-native, typically synthetic, metal cofactors, are a flourishing class of biocatalyst for unnatural reactions. Although the number of these reactions is rapidly increasing, multi-faceted mechanistic studies of ArMs comprising structural, kinetic, computational and cofactor binding data to reveal detailed mechanistic information on the effects of the protein scaffold on the structure and reactivity of ArMs are more limited. Here we report the structure of an unnatural P450 analogue using X-ray diffraction. We also report the kinetic analysis of its reaction, catalyst activation during an induction period, and the origins of the stereoselectivity for the cyclopropanation of a terpene catalysed by the iridium-containing P450 variant (Ir(Me)-CYP119). Our data reveal a mechanism initiated by the flip of the cofactor from an inactive to an active conformation. This change in conformation is followed by thousands of turnovers occurring by rate-determining formation of an iridium-carbene intermediate, thereby highlighting the influence of cofactor dynamics within a single active site on an ArM-catalysed reaction.
人工金属酶（ArMs）含有非天然的、通常是合成的金属辅因子，是一类用于非天然反应的蓬勃发展的生物催化剂。尽管这些反应的数量在迅速增加，但对ArMs进行的多方面机理研究，包括结合结构、动力学、计算和辅因子结合数据，以揭示有关蛋白质支架对ArMs结构和反应性影响的详细机理信息，却较为有限。在此，我们报道了一种非天然P450类似物的X射线衍射结构。我们还报告了其反应的动力学分析、诱导期的催化剂活化以及含铱P450变体（Ir(Me)-CYP119）催化的萜烯环丙烷化反应的立体选择性起源。我们的数据揭示了一种由辅因子从无活性构象转变为活性构象引发的机制。这种构象变化之后是通过铱-卡宾中间体的速率决定形成发生数千次周转，从而突出了单个活性位点内辅因子动力学对ArM催化反应的影响。
