Engineering of a P450-based Kemp eliminase with a new mechanism

doi:10.1016/S1872-2067(23)64389-X

Abstract

Abstract:

For three decades the biocatalytic version of the Kemp elimination of 5-nitro-benzisoxazole (1) has served as a forum for testing the creation of different artificial enzymes, the primary aim being to reveal mechanistic intricacies and to extend our understanding of enzymatic catalysis as such. In general, acid/base catalysis pertains, but recently a novel redox based mechanism was postulated when using P450-BM3 mutants as scaffolds. In the present study, we report an surprising discovery made upon employing new P450-BM3 variants generated by rational enzyme design, which points to the existence of a new and different redox based mechanism. X-ray structural data and theoretical analyses based on MD simulations and QM/MM calculations support this conclusion. The results of this study are of relevance in the human metabolism of therapeutic drugs and in redox mediated biosynthesis catalyzed by P450s.

Key words: Kemp elimination, Redox mechanism, P450 monooxygenase, Quantum mechanical/molecular, mechanical, X-ray structure, Saturation mutagenesis

Aitao Li, Qian Wang, Xitong Song, Xiaodong Zhang, Jian-Wen Huang, Chun-Chi Chen, Rey-Ting Guo, Binju Wang, Manfred T. Reetz. Engineering of a P450-based Kemp eliminase with a new mechanism[J]. Chinese Journal of Catalysis, 2023, 47: 191-199.

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URL: https://www.cjcatal.com/EN/10.1016/S1872-2067(23)64389-X

https://www.cjcatal.com/EN/Y2023/V47/I4/191

Figures/Tables 8

Fig. 1. Designed enzyme catalyzed Kemp elimination via a Br?nsted acid/base mechanism (B = base; BH = acid) [16].

Fig. 2. Redox based P450-BM3 catalyzed Kemp elimination via two different mechanisms. (a) Previously reported P450-BM3 catalyzed Kemp elimination with a redox-based mechanism [16]. (b) New mechanism of a redox based Kemp eliminase using rationally selected mutants of P450-BM3.

Fig. 3. Engineering of P450-BM3 for Kemp elimination of substrate 1. (a) Zoom of the active site of WT P450-BM3. (b) The present strategy of directed evolution for P450-BM3 with F87G as template. Double code saturation mutagenesis (DCSM) at two randomization sites A (S72, L75, T438) and B (A82, L181, A328), using bulky lysine and tyrosine as the double code K-Y. (c) Michaelis-Menten plots for triple mutant F87G/L75Y/T438K (TMK) (red) and WT (blue). The data represents the average of three independent measurements, with error bars denoting s.d.

Table 1 Summary of kinetic parameters for P450-BM3 and variants catalyzing the Kemp elimination of 1 with different mechanisms. In all cases purified enzymes were used a.

Catalyst	K_m (mmol L^‒1)	k_cat (s^-1)	k_cat/K_m (L s^-1 mol^-1)		Reference
WT P450BM-3	>6	>1.5	240 ± 60	N/A	[17]
F87G	2.5 ± 0.6	3.0 ± 0.5	1,200 ± 200	2.6 × 10⁶
A82F	0.27±0.03	8.4 ± 0.4	31000 ± 1,500	0.7 × 10⁷
F87G/A82F	1.3 ± 0.2	11.5 ± 0.7	8800 ± 700	1.0 × 10⁷
F87G/L75Y	1.7 ± 0.2	4.4 ± 0.2	2600 ± 100	3.8 × 10⁶	This study
F87G/L75Y/T438K	4.7 ± 0.5	27.4 ± 2.2	5800 ± 400	2.4 × 10⁷
F87G/L75Y/T438R	6.5 ± 0.9	28.1 ± 3.3	4300 ± 500	2.4 × 10⁷
F87G/L75Y/T438L	1.6 ± 0.2	9.0 ± 0.2	5600 ± 100	0.8 × 10⁷

Fig. 4. Exploration of deconvoluted mutations in TMK (F87G/L75Y/T438K) as catalysts in the Kemp elimination of substrate 1. (a) Kemp elimination of substrate 1 catalyzed by the P450-BM3 variant TMK (F87G/L75Y/T438K) and by deconvoluted variants thereof with the cell free extract. (b) Kemp elimination of substrate 1 catalyzed by P450-BM3 variants with different amino acid substitutions at position 438 based on the parental variant F87G/L75Y; cell free extracts were used as catalysts.

Fig. 5. Crystal structure of apo- and ligand-bound form of P450-BM3 variant TMK. (a) The overall structure of each crystal is presented in cartoon models, with heme and bound ligands shown in sticks. (b) The protein-ligand interaction networks in TMK-substrate complex. Hemes are shown by purple blue sticks and soaked ligands in tint/magenta sticks. Amino acids are shown in lines, with mutated ones colored in the same scheme as the bound ligands. The yellow dashed lines indicate distance < 3.5 ?.

Fig. 6. Illustration of MD analyses. (a) Non-bonding interactions between the substrate and the protein environment obtained from 50 ns MD simulations. The red line corresponds to the binding conformation in which the nitro group is in close proximity to heme-Fe (Fig. 6(b)), with an average non-bonding interaction value of ?30.8 kcal mol?1. The black line corresponds to the binding conformation in which the N1 atom of substrate is in close proximity to heme-Fe (Fig. 6(c)), with an average non-bonding interaction value of ?26.5 kcal mol?1. (b) The most populated snapshot from clustering of MD trajectory for the substrate binding conformation in which the nitro group is in close proximity to heme-Fe. (c) The most populated snapshot from clustering of MD trajectory for the substrate binding conformation in which the N1 atom of substrate is in close proximity to heme-Fe.

Fig. 7. (a) The calculated mechanism for the TMK-catalyzed Kemp elimination derived from QM/MM simulations. (b) The representative structures of key species obtained from QM/MM simulations (key distances are given in angstroms) (Fig. S9). The spin densities of iron and substrate are given in blue font.

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