Flexibility Regulation of Loops Surrounding the Tunnel Entrance in Cytochrome P450 Enhanced Substrate Access Substantially

Li, Zhongyu; Meng, Shuaiqi; Nie, Kaili; Schwaneberg, Ulrich; Davari, Mehdi D.; Xu, Haijun; Ji, Yu; Liu, Luo

doi:10.1021/acscatal.2c02258

Cited by 42 publications

(41 citation statements)

References 49 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Therefore, we reasoned that the peroxygenase activity of P450s could be improved by engineering some of the water tunnels on the basis of the reported tunnel engineering strategy to ensure suitable inflow pathways for water-soluble H 2 O 2 (Figure ). In this study, we have designed and broadened the water tunnels of P450s to facilitate the H 2 O 2 flow to the active site. These modifications significantly increase the peroxygenase activity of three P450 enzymes, including NADH-dependent CYP199A4 and CYP153A M.aq , as well as DFSM-facilitated P450BM3 peroxygenase.…”

Section: Introductionmentioning

confidence: 99%

Enabling Peroxygenase Activity in Cytochrome P450 Monooxygenases by Engineering Hydrogen Peroxide Tunnels

et al. 2023

View full text Add to dashboard Cite

Given prominent physicochemical similarities between H2O2 and water, we report a new strategy for promoting the peroxygenase activity of P450 enzymes by engineering their water tunnels to facilitate H2O2 access to the heme center buried therein. Specifically, the H2O2-driven activities of two native NADH-dependent P450 enzymes (CYP199A4 and CYP153A M.aq ) increase significantly (by >183-fold and >15-fold, respectively). Additionally, the amount of H2O2 required for an artificial P450 peroxygenase facilitated by a dual-functional small molecule to obtain the desired product is reduced by 95%–97.5% (with ∼95% coupling efficiency). Structural analysis suggests that mutating the residue at the bottleneck of the water tunnel may open a second pathway for H2O2 to flow to the heme center (in addition to the natural substrate tunnel). This study highlights a promising, generalizable strategy whereby P450 monooxygenases can be modified to adopt peroxygenase activity through H2O2 tunnel engineering, thus broadening the application scope of P450s in synthetic chemistry and synthetic biology.

show abstract

Section: Introductionmentioning

confidence: 99%

Enabling Peroxygenase Activity in Cytochrome P450 Monooxygenases by Engineering Hydrogen Peroxide Tunnels

et al. 2023

View full text Add to dashboard Cite

show abstract

“…Tunnel analyses including throughput, tunnel curvature, length, and substrate were introduced to explore the improved catalytic efficiency. 47 The geometric properties of the tunnel were prominently analyzed to reveal the ligand transport process. The results showed that the average curvature of PaAmi M1…”

Section: Static and Dynamic Tunnel Analysis Of Paami M1 And Its Tripl...mentioning

confidence: 99%

Structure-Oriented Engineering of Amidase: Modification of Twisted Access Tunnel for Efficient Synthesis of 2-Chloronicotinic Acid

Xie

Zhang

et al. 2023

ACS Catal.

View full text Add to dashboard Cite

Amidases are robust biocatalysts for the industrial synthesis of carboxylic acids. Amidase-catalyzed hydrolysis of 2chloronicotinamide is regarded as a promising route for 2-chloronicotinic acid production, which is an important building block for agrochemicals and pharmaceuticals. However, the o-substituted Cl of the pyridine ring caused a strong electronic effect and steric hindrance. Engineering access tunnels is one of the most efficient strategies to improve catalytic efficiency by adjusting the transfer efficiency of ligands. Herein, we engineered a twisted access tunnel of amidase and the mutant PaAmi M1A378V+V402L+L403V was achieved, which exhibited increased catalytic efficiency toward 2-chloronicotinamide. Tunnel analysis, molecular dynamics simulations, and quantum mechanical calculations revealed that the straightened tunnel significantly improved the transport efficiency and simultaneously facilitated substrate binding and nucleophilic attack. With the best mutant as a biocatalyst, 1330 mM product was accumulated within 2 h and a high space-time-yield of 2515 g product L −1 d −1 was achieved, which is the highest ever reported. These results lay the foundation for industrial production of 2-chloronicotinic acid and give important guidance for engineering amidases into versatile biocatalysts.

show abstract

“…Similarly, highly flexible loops surrounding the tunnel entrance of a cytochrome P450 were engineered to enhance its substrate access. The best variant obtained reduced the flexibility of two critical loops, responsible for improving the stability of the substrate access tunnel, and showed 134-fold improvement in the catalytic activity ( Li Z. et al, 2022 ). A loop engineering strategy was also applied to improve the activity and selectivity of a cumene dioxygenase from Pseudomonas fluorescens IP01.…”

Section: Rational Design Strategy Based On Dynamics Modificationmentioning

confidence: 99%

Rational design of enzyme activity and enantioselectivity

Song

Zhang

Wu³

et al. 2023

Front. Bioeng. Biotechnol.

View full text Add to dashboard Cite

The strategy of rational design to engineer enzymes is to predict the potential mutants based on the understanding of the relationships between protein structure and function, and subsequently introduce the mutations using the site-directed mutagenesis. Rational design methods are universal, relatively fast and have the potential to be developed into algorithms that can quantitatively predict the performance of the designed sequences. Compared to the protein stability, it was more challenging to design an enzyme with improved activity or selectivity, due to the complexity of enzyme molecular structure and inadequate understanding of the relationships between enzyme structures and functions. However, with the development of computational force, advanced algorithm and a deeper understanding of enzyme catalytic mechanisms, rational design could significantly simplify the process of engineering enzyme functions and the number of studies applying rational design strategy has been increasing. Here, we reviewed the recent advances of applying the rational design strategy to engineer enzyme functions including activity and enantioselectivity. Five strategies including multiple sequence alignment, strategy based on steric hindrance, strategy based on remodeling interaction network, strategy based on dynamics modification and computational protein design are discussed and the successful cases using these strategies are introduced.

show abstract

Flexibility Regulation of Loops Surrounding the Tunnel Entrance in Cytochrome P450 Enhanced Substrate Access Substantially

Cited by 42 publications

References 49 publications

Enabling Peroxygenase Activity in Cytochrome P450 Monooxygenases by Engineering Hydrogen Peroxide Tunnels

Enabling Peroxygenase Activity in Cytochrome P450 Monooxygenases by Engineering Hydrogen Peroxide Tunnels

Structure-Oriented Engineering of Amidase: Modification of Twisted Access Tunnel for Efficient Synthesis of 2-Chloronicotinic Acid

Rational design of enzyme activity and enantioselectivity

Contact Info

Product

Resources

About