Introduction of aromatic amino acids in electron transfer pathways yielded improved catalytic performance of cytochrome P450s

doi:10.1016/S1872-2067(23)64445-6

Abstract

Abstract:

Cytochrome P450s are versatile catalysts for biosynthesis applications. In the P450 catalytic cycle, two electrons are required to reduce the heme iron and activate the subsequent reductions through proposed electron transfer pathways (eTPs), which often represent the rate-limiting step in reactions. Herein, the P450 BM3 from Bacillus megaterium was engineered for improved catalytic performance by redesigning proposed eTPs. By introducing aromatic amino acids on eTPs of P450 BM3, the “best” variant P2H02 (A399Y/Q403F) showed 13.9-fold improved catalytic efficiency (k_cat/K_M = 913.5 L mol^‒1 s^‒1) compared with P450 BM3 WT (k_cat/K_M = 65.8 L mol^‒1 s^‒1). Molecular dynamics simulations and electron hopping pathways analysis revealed that aromatic amino acid substitutions bridging the cofactor flavin mononucleotide and heme iron could increase electron transfer rates and improve catalytic performance. Moreover, the introduction of tyrosines showed positive effects on catalytic efficiency by potentially protecting P450 from oxidative damage. In essence, engineering of eTPs by aromatic amino acid substitutions represents a powerful approach to design catalytically efficient P450s (such as CYP116B3) and could be expanded to other oxidoreductases relying on long-range electron transfer pathways.

Key words: Directed evolution, P450 BM3, Protein engineering, Electron transfer, Rational design

Shuaiqi Meng, Zhongyu Li, Yu Ji, Anna Joelle Ruff, Luo Liu, Mehdi D. Davari, Ulrich Schwaneberg. Introduction of aromatic amino acids in electron transfer pathways yielded improved catalytic performance of cytochrome P450s[J]. Chinese Journal of Catalysis, 2023, 49: 81-90.

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URL: https://www.cjcatal.com/EN/10.1016/S1872-2067(23)64445-6

https://www.cjcatal.com/EN/Y2023/V49/I6/81

Figures/Tables 5

Fig. 1. The selected residues on putative eTPs between cofactor FMN and heme iron of P450 BM3 (PDB ID: 1BVY (16)) for SSM. The FMN and heme domains are shown in the orange and green cartoons, respectively. The targeted amino acid residues are shown as cyan sticks.

Table 1 The kinetic parameters, coupling efficiency, and total turnover number (TTN) of P450 BM3 WT and seven improved variants from TDB library. The values were obtained from triplicate measurements. P1H11: F390Y/A399Y/Q403F; P1G03: F390Y/A399F/Q403Y; P1H02: A399F/Q403F; P1H09: A399F/Q403Y; P2H02: A399Y/Q403F; P2G03: F390Y/A399F/Q403F; P3A04: F390Y/A399F/Q403W.

Variant	K_M (mmol L^‒1)	k_cat (s^‒1)	k_cat/K_M (L mol^‒1 s^‒1)	Coupling efficiency	TTN*
WT	10.95 ± 0.80	0.72 ± 0.05	65.8	31.18 ± 0.97	1196 ± 70
P1H11 (F390Y/A399Y/Q403F)	2.31 ± 0.18	1.13 ± 0.10	489.2	48.25 ± 1.53	1984 ± 27
P1G03 (F390Y/A399F/Q403Y)	3.26 ± 0.38	1.18 ± 0.01	362.0	47.41 ± 0.39	2332 ± 131
P1H02 (A399F/Q403F)	8.61 ± 0.16	1.14 ± 0.06	132.4	47.92 ± 1.22	2560 ± 28
P1H09 (A399F/Q403Y)	6.51 ± 0.28	1.28 ± 0.03	196.6	49.81 ± 2.99	2207 ± 62
P2H02 (A399Y/Q403F)	1.04 ± 0.21	0.95 ± 0.03	913.5	47.61 ± 2.50	2582 ± 108
P2G03 (F390Y/A399F/Q403F)	1.66 ± 0.51	1.14 ± 0.01	686.7	44.33 ± 1.68	2615 ± 85
P3A04 (F390Y/A399F/Q403W)	3.21 ± 0.88	1.21 ± 0.09	376.9	37.97 ± 1.42	2174 ± 70

Fig. 2. The superexchange efficiencies between the cofactor FMN and heme iron of P450 BM3 WT and improved variants from TDB library. (a) The edge-to-edge distances between the cofactor FMN and heme iron determined from 50 ns MD simulations. The MD simulations were run in triplicate (run_1, run_2, run_3) and shown with orange, brown, and blue lines, respectively. (b) The time-averaged distances between the cofactor FMN and heme iron (blue column) and corresponding ET rate (orange scatter) of P450 BM3 WT and beneficial variants. The distances were averaged over the last 40 ns from three independent MD runs. The ET rates were calculated by equation (2) utilizing the time-averaged distance. (c) Comparison of total turnover number (TTN) and the time-averaged distance between the cofactor FMN and heme iron of P450 WT and beneficial variants. The dashed line shows the linear fit to the scattered points. (d) Comparison of coupling efficiency and the time-averaged distance between the cofactor FMN and heme iron of P450 WT and beneficial variants. The dashed line shows the linear fit to the scattered points.

Fig. 3. Predicted electron hopping pathways between the cofactor FMN and heme iron of P450 BM3: P450 BM3 WT (a) and variant P2H02 (b). The results are visualized in 3D (left) and 2D (right). The electron hopping directions are shown in black arrows. In 2D visualization, the center-of-mass distances (in ?) between neighboring residues are shown in purple.

Fig. 4. Proposed rescue pathway of P450 BM3 and variant P2H02. (a) The schematic diagram of oxidative damage of P450 BM3. The red explosions repent the potential oxidation compound. The probable antioxidant rescue pathways from the cofactor heme to the surface of P450 BM3 WT (b) and variant P2H02 (c). The pathways of P450 BM3 were indicated by colors in grey, and the new pathway in variant P2H02 was indicated by colors in orange. The arrows showed the hole transport directions. τM represented the exact mean residence time, which is defined as the inverse of the effective start-to-finish transfer rate [39]. Low τM value represents the fast hole hopping rate.

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