Discovery of a P450-Catalyzed Oxidative Defluorination Mechanism toward Chiral Organofluorines: Uncovering a Hidden Pathway

Huang, Qi; Zhang, Xuan; Chen, Qianqian; Tian, Shaixiao; Wei, Tong; Zhang, Wei; Chen, Yingzhuang; Ma, Ming; Chen, Bo; Wang, Binju; Wang, Jianbo

doi:10.1021/acscatal.1c05510

Cited by 12 publications

(5 citation statements)

References 67 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The abovementioned double-mutant DN46 was constructed by sitedirected mutagenesis. In our previous work, 37 we had found that the wild type (WT) of this enzyme catalyzes the reduction of ethyl-2-oxo-2-phenylacetate, which motivated us to explore its general utilization for asymmetric synthesis by directed evolution. Therefore, optimization of the reaction conditions is necessary before performing protein engineering.…”

Section: ■ Resultsmentioning

confidence: 99%

From Semirational to Rational Design: Developing a Substrate-Coupled System of Glucose Dehydrogenase for Asymmetric Synthesis

et al. 2022

Self Cite

View full text Add to dashboard Cite

Glucose dehydrogenase (GDH) generally functions as an expensive cofactor NAD(P)H regenerator in an enzyme-coupled cofactor regeneration system, as in the production of fine chemicals. However, whether GDH can accept substrates other than glucose remains to be explored. Based on a known mutant of GDH with high thermostability, DN46 (E170K–K252L), we employed semirational design-based directed evolution to expand its substrate scope and promote its application in the asymmetric synthesis of methyl 2-hydroxyl carboxylates. After one round of saturation mutagenesis and two rounds of iterative saturation mutagenesis, an enantioselectivity-enhanced mutant DN46-E96Q-H147V (>99% ee, R-preference) and enantioselectivity-reversed mutant DN46-E96Q-I150A-W152L with high enantioselectivity (>97% ee, S-preference) evolved. With glucose as a cosubstrate, we developed a dual-function GDH-based substrate-coupled cofactor regeneration system for asymmetric synthesis. Furthermore, in light of a deeper understanding of the catalytic mechanism, a rational design was successfully performed to create a mutant DN46-W152G for the upscaled synthesis of (R)-2-chloromandelic acid methyl ester, the precursor of the medicine (S)-clopidogrel. This work expands the utilization of GDH, provides a design method for rational design of this enzyme, and will support future work regarding its application toward achieving even broader substrate scope.

show abstract

Section: ■ Resultsmentioning

confidence: 99%

From Semirational to Rational Design: Developing a Substrate-Coupled System of Glucose Dehydrogenase for Asymmetric Synthesis

et al. 2022

Self Cite

View full text Add to dashboard Cite

show abstract

“…The corrections between the stable structures and the transition states were ascertained by analyzing the corresponding imaginary frequency modes, as well as by limited intrinsic reaction coordinate (IRC) calculations. The energies were further refined at BMK/6-311 + +G(d,p) 73 , which has been proven to be reliable for HCC model calculations [74][75][76] .…”

Section: Qm Calculationsmentioning

confidence: 99%

Endowing homodimeric carbamoyltransferase GdmN with iterative functions through structural characterization and mechanistic studies

et al. 2022

Self Cite

View full text Add to dashboard Cite

Iterative enzymes, which catalyze sequential reactions, have the potential to improve the atom economy and diversity of industrial enzymatic processes. Redesigning one-step enzymes to be iterative biocatalysts could further enhance these processes. Carbamoyltransferases (CTases) catalyze carbamoylation, an important modification for the bioactivity of many secondary metabolites with pharmaceutical applications. To generate an iterative CTase, we determine the X-ray structure of GdmN, a one-step CTase involved in ansamycin biosynthesis. GdmN forms a face-to-face homodimer through unusual C-terminal domains, a previously unknown functional form for CTases. Structural determination of GdmN complexed with multiple intermediates elucidates the carbamoylation process and identifies key binding residues within a spacious substrate-binding pocket. Further structural and computational analyses enable multi-site enzyme engineering, resulting in an iterative CTase with the capacity for successive 7-O and 3-O carbamoylations. Our findings reveal a subclade of the CTase family and exemplify the potential of protein engineering for generating iterative enzymes.

show abstract

“…It has been the topic of extensive research over the past three decades and numerous laboratories have worked on evolving this enzyme to generate novel enzymatic properties and features. As a result, P450 BM3 and its variants have been shown to catalyze a wide variety of reactions, including hydroxylation, − epoxidation, − carbene transfer, − and nitrene transfer, − with a diverse range of substrates. The advances reported by the group of Frances Arnold on the evolution of P450 BM3 toward novel biocatalytic reactions, which were prominently featured in her 2018 Nobel-winning research, are among the most transformative.…”

Section: Introductionmentioning

confidence: 99%

Choose Your Own Adventure: A Comprehensive Database of Reactions Catalyzed by Cytochrome P450 BM3 Variants

Fansher,

Besna,

Fendri

et al. 2024

ACS Catal.

View full text Add to dashboard Cite

Cytochrome P450 BM3 monooxygenase is the topic of extensive research as many researchers have evolved this enzyme to generate a variety of products. However, the abundance of information on increasingly diversified variants of P450 BM3 that catalyze a broad array of chemistry is not in a format that enables easy extraction and interpretation. We present a database that categorizes variants by their catalyzed reactions and includes details about substrates to provide reaction context. This database of >1500 P450 BM3 variants is downloadable and machine-readable and includes instructions to maximize ease of gathering information. The database allows rapid identification of commonly reported substitutions, aiding researchers who are unfamiliar with the enzyme in identifying starting points for enzyme engineering. For those actively engaged in engineering P450 BM3, the database, along with this review, provides a powerful and user-friendly platform to understand, predict, and identify the attributes of P450 BM3 variants, encouraging the further engineering of this enzyme.

show abstract

Discovery of a P450-Catalyzed Oxidative Defluorination Mechanism toward Chiral Organofluorines: Uncovering a Hidden Pathway

Cited by 12 publications

References 67 publications

From Semirational to Rational Design: Developing a Substrate-Coupled System of Glucose Dehydrogenase for Asymmetric Synthesis

From Semirational to Rational Design: Developing a Substrate-Coupled System of Glucose Dehydrogenase for Asymmetric Synthesis

Endowing homodimeric carbamoyltransferase GdmN with iterative functions through structural characterization and mechanistic studies

Choose Your Own Adventure: A Comprehensive Database of Reactions Catalyzed by Cytochrome P450 BM3 Variants

Contact Info

Product

Resources

About