Mechanism-Guided Design and Discovery of Efficient Cytochrome P450-Derived C–H Amination Biocatalysts

Abstract

Cytochromes P450 have been recently identified as a promising class of biocatalysts for mediating C–H aminations via nitrene transfer, a valuable transformation for forging new C–N bonds. The catalytic efficiency of P450s in these non-native transformations is however significantly inferior to that exhibited by these enzymes in their native monooxygenase function. Using a mechanism-guided strategy, we report here the rational design of a series of P450BM3-based variants with dramatically enhanced C–H amination activity acquired through disruption of the native proton relay network and other highly conserved structural elements within this class of enzymes. This approach further guided the identification of XplA and BezE, two “atypical” natural P450s implicated in the degradation of a man-made explosive and in benzastatins biosynthesis, respectively, as very efficient C–H aminases. Both XplA and BezE could be engineered to further improve their C–H amination reactivity, which demonstrates their evolvability for abiological reactions. These engineered and natural P450 catalysts can promote the intramolecular C–H amination of arylsulfonyl azides with over 10 000–14 000 catalytic turnovers, ranking among the most efficient nitrene transfer biocatalysts reported to date. Mechanistic and structure–reactivity studies provide insights into the origin of the C–H amination reactivity enhancement and highlight the divergent structural requirements inherent to supporting C–H amination versus C–H monooxygenation reactivity within this class of enzymes. Overall, this work provides new promising scaffolds for the development of nitrene transferases and demonstrates the value of mechanism-driven rational design as a strategy for improving the catalytic efficiency of metalloenzymes in the context of abiological transformations.

Combining chemistry and protein engineering for new-to-nature biocatalysis

“…inquiries of Breslow and Gellman into enzymatic nitrene transfer, engineered enzymes now perform highly stereoselective C-H amination reactions with TTN values of hundreds to thousands (Fig. 1c) [24][25][26][27] . The mechanistic analogy between biomimetic iron porphyrin catalysts and haemoproteins led to the discovery of other nonnatural reactivities supported by these proteins, such as carbene transfer, and even haemoproteins with no natural enzyme functions (for example, globins and cytochrome c) have been engineered for abiological carbene-transfer activity (Fig.…”

“…3c) 26 . In this system, The chemistry of metal porphyrins has been extended to haemoproteins for both carbene and nitrene transfer when presented with carbene and nitrene precursors [24][25][26][27][28][29][30][31] . X and r are shown in Fig.…”

Exploring and Adapting the Molecular Selectivity of Artificial Metalloenzymes

“…With the number of literature examples growing constantly, these new-to-nature reactions have included aldehyde olefination, [14][15] alkyne cyclopropenation, [16][17] alkene cyclopropanation, [18][19][20][21][22][23][24][25][26][27][28][29][30][31][32][33][34][35][36] the Doyle-Kirmse reaction, 37 N−H bond insertion, [38][39][40] S−H bond insertion, 27,41 Si−H bond insertion, [42][43] B−H bond insertion, [44][45][46] and C-H insertion. [47][48][49] Other works have also shown high enantioselectivity with C(sp 3 )−H bond aminations that can proceed either intramolecularly, 36,[50][51][52][53][54][55][56][57] or intermolecularly. [58][59]<...>…”

“…RI18, both of which were shown to facilitate high rates of intramolecular C−H bond aminations. 57 By far one of the most impressive concepts to derive from studying P450s has been the work involving cytochrome "P411"s. 18 By replacing the native P450 cysteine-heme axial ligation with a unique serine-heme ligation (via C400S), these artificial P411s thus gained enhanced reactivity for a number of carbene transfer reactions. 16-17, 22, 27-28, 30, 50-51, 53, 58-60, 62-63 The theory behind this approach is that the reduction potential for the resting state of Fe III -to-Fe II will become higher than that of NAD(P) + -to-NAD(P)H, thereby driving the efficient production of the catalytic Fe II state.…”

The molecular mechanism of P450-catalyzed amination of the pyrrolidine derivative of lidocaine: insights from multiscale simulations

“…Notably, a few P450 oxidases have been discovered to catalyse the intra-or inter-molecular C-N bondforming reactions in recent studies. [15][16][17][18][19][20] It was suggested 14 that the reaction is initiated by the Cpd I-mediated H-atom abstraction (HAA) from the pyrrolidine derivative of lidocaine, leading to substrate radical and Fe(IV)-OH intermediate (Scheme 1). Next, two plausible mechanisms were proposed for P450-catalyzed amination of the pyrrolidine derivative of lidocaine.…”