Engineering the biomimetic cofactors of NMNH for cytochrome P450 BM3 based on binding conformation refinement

Liu, Yao; Cong, Yalong; Zhang, Chuan‐Xi; Fang, Bohuan; Pan, Yiou; Li, Qiangzi; You, Chun; Gao, Bei; Qi, Yifei; Zhu, Tong; Zhang, Lujia

doi:10.1039/d1ra00352f

Cited by 5 publications

(2 citation statements)

References 35 publications

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“…The lack of clear structural basis for improved activities can be aided with MD analysis. Liu et al 47 performed virtual screening with computational binding affinity predictions and MD analysis of binding pose stability to select mutant P450-BM3 using NMNH. Their pipeline led to identification of P450-BM3 S848R with ~2-fold increase in NMNH activity over the wild type.…”

Section: Discussionmentioning

confidence: 99%

Orthogonal glycolytic pathway enables directed evolution of noncanonical cofactor oxidase

et al. 2022

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Noncanonical cofactor biomimetics (NCBs) such as nicotinamide mononucleotide (NMN+) provide enhanced scalability for biomanufacturing. However, engineering enzymes to accept NCBs is difficult. Here, we establish a growth selection platform to evolve enzymes to utilize NMN+-based reducing power. This is based on an orthogonal, NMN+-dependent glycolytic pathway in Escherichia coli which can be coupled to any reciprocal enzyme to recycle the ensuing reduced NMN+. With a throughput of >106 variants per iteration, the growth selection discovers a Lactobacillus pentosus NADH oxidase variant with ~10-fold increase in NMNH catalytic efficiency and enhanced activity for other NCBs. Molecular modeling and experimental validation suggest that instead of directly contacting NCBs, the mutations optimize the enzyme’s global conformational dynamics to resemble the WT with the native cofactor bound. Restoring the enzyme’s access to catalytically competent conformation states via deep navigation of protein sequence space with high-throughput evolution provides a universal route to engineer NCB-dependent enzymes.

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Section: Discussionmentioning

confidence: 99%

Orthogonal glycolytic pathway enables directed evolution of noncanonical cofactor oxidase

et al. 2022

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“…The specific activity of aMOx and mutants was determined by evaluating the consumption of NADPH ( Liu et al, 2021 ). The Bradford (1976) method was used to determine the protein concentration using a Bradford protein quantification kit (YEASEN Biotechnology Co., Ltd., Shanghai, China).…”

Section: Methodsmentioning

confidence: 99%

Rational Design of P450 aMOx for Improving Anti-Markovnikov Selectivity Based on the “Butterfly” Model

Pan

Bao

Zhang

et al. 2022

Front. Mol. Biosci.

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Aromatic aldehydes are important industrial raw materials mainly synthesized by anti-Markovnikov (AM) oxidation of corresponding aromatic olefins. The AM product selectivity remains a big challenge. P450 aMOx is the first reported enzyme that could catalyze AM oxidation of aromatic olefins. Here, we reported a rational design strategy based on the “butterfly” model of the active site of P450 aMOx. Constrained molecular dynamic simulations and a binding energy analysis of key residuals combined with an experimental alanine scan were applied. As a result, the mutant A275G showed high AM selectivity of >99%. The results also proved that the “butterfly” model is an effective design strategy for enzymes.

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Stoichiometric Regeneration of Biomimetic Nicotinamide Coenzyme Powered by Biomass Sugars via In Vitro Synthetic Enzymatic Biosystems

Li,

Su,

Meng

et al. 2024

ChemSusChem

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Biomimetic nicotinamide coenzymes, including nicotinamide mononucleotide (NMN+), have been demonstrated as promising low‐cost alternatives to nicotinamide adenine dinucleotide (phosphate) (NAD(P)+) in biocatalysis. Herein, to efficiently regenerate NMNH from NMN+ in vitro powered by biomass sugars, a thermophilic NADP+‐dependent glucose 6‐phosphate dehydrogenase from Thermotoga maritima (TmG6PDH) was engineered to increase the activity toward NMN+. The catalytic efficiency (kcat/Km) of optimal mutant (TmG6PDH‐R7) toward NMN+ increased by 71.7‐fold than TmG6PDH‐WT. As a result, compared to the wild type, the coenzyme specificity ([kcat/Km]NMN+/[kcat/Km]NADP+) of TmG6PDH‐R7 increased by ~2.0×105‐fold. The structural analysis revealed that the introduced hydrophobic and bulky residues lead to the formation of a smaller binding pocket, which resulting in a higher affinity for NMN+ with small size than NADP+. Then several in vitro synthetic enzymatic biosystems (ivSEBs) comprising this thermophilic TmG6PDH‐R7 and a previously engineered thermophilic 6‐phosphogluconate dehydrogenase were constructed. These ivSEBs harnessed the complete oxidation of renewable biomass sugars to facilitate the stoichiometric regeneration of 12 molecules of NMNH from 1 molecule of glucose, thereafter producing various products such as levodione, 2,3‐butanediol or bioelectricity, over a wide temperature range. This study could pave the way for using stable and low‐cost biomimetic coenzymes in ivSEBs for industrial biomanufacturing.

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Engineering the biomimetic cofactors of NMNH for cytochrome P450 BM3 based on binding conformation refinement

Abstract: A rational design strategy was proposed to improve the efficient utilization of alternative biomimetic cofactor by P450 BM3 enzyme.

Cited by 5 publications

References 35 publications

Orthogonal glycolytic pathway enables directed evolution of noncanonical cofactor oxidase

Orthogonal glycolytic pathway enables directed evolution of noncanonical cofactor oxidase

Rational Design of P450 aMOx for Improving Anti-Markovnikov Selectivity Based on the “Butterfly” Model

Stoichiometric Regeneration of Biomimetic Nicotinamide Coenzyme Powered by Biomass Sugars via In Vitro Synthetic Enzymatic Biosystems

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