ArticleChembiochem : a European journal of chemical biology2014
Enhancing the efficiency and regioselectivity of P450 oxidation catalysts by unnatural amino acid mutagenesis.
Article in Chembiochem : a European journal of chemical biology, 2014. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 33 papers.
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33 citing papers in PubMed.
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- Noncanonical Amino Acids: Bringing New-to-Nature Functionalities to Biocatalysis.Chemical reviews · 2024Review
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- Noncanonical Amino Acids in Biocatalysis.Chemical reviews · 2024Review
- Choose Your Own Adventure: A Comprehensive Database of Reactions Catalyzed by Cytochrome P450 BM3 Variants.ACS catalysis · 2024Review
- Artificial Small Molecules as Cofactors and Biomacromolecular Building Blocks in Synthetic Biology: Design, Synthesis, Applications, and Challenges.Molecules (Basel, Switzerland) · 2023Review
- Unnatural activities and mechanistic insights of cytochrome P450 PikC gained from site-specific mutagenesis by non-canonical amino acids.Nature communications · 2023Article
- Enantioselective Single and Dual α-C-H Bond Functionalization of Cyclic Amines via Enzymatic Carbene Transfer.Journal of the American Chemical Society · 2023Article
- Non-Canonical Amino Acid-Based Engineering of (Frontiers in chemistry · 2022Article
- Reprogramming natural proteins using unnatural amino acids.RSC advances · 2021Review
- Recent advancements in enzyme engineering via site-specific incorporation of unnatural amino acids.World journal of microbiology & biotechnology · 2021Review
- Introducing noncanonical amino acids for studying and engineering bacterial microcompartments.Current opinion in microbiology · 2021Review
- Comprehensive Structure-Activity Profiling of Micheliolide and its Targeted Proteome in Leukemia Cells via Probe-Guided Late-Stage C-H Functionalization.ACS central science · 2021Article
- Scalable biocatalytic C-H oxyfunctionalization reactions.Chemical Society reviews · 2020Review
- Folding Assessment of Incorporation of Noncanonical Amino Acids Facilitates Expansion of Functional-Group Diversity for Enzyme Engineering.Chemistry (Weinheim an der Bergstrasse, Germany) · 2020Article
- Mechanism-Guided Design and Discovery of Efficient Cytochrome P450-Derived C-H Amination Biocatalysts.Journal of the American Chemical Society · 2020Article
- The Hitchhiker's guide to biocatalysis: recent advances in the use of enzymes in organic synthesis.Chemical science · 2020Review
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4 authors.
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Abstract
The development of effective strategies for modulating the reactivity and selectivity of cytochrome P450 enzymes represents a key step toward expediting the use of these biocatalysts for synthetic applications. We have investigated the potential of unnatural amino acid mutagenesis to aid efforts in this direction. Four unnatural amino acids with diverse aromatic side chains were incorporated at 11 active-site positions of a substrate-promiscuous CYP102A1 variant. The resulting "uP450s" were then tested for their catalytic activity and regioselectivity in the oxidation of two representative substrates: a small-molecule drug and a natural product. Large shifts in regioselectivity resulted from these single mutations, and in particular, for para-acetyl-Phe substitutions at positions close to the heme cofactor. Screening this mini library of uP450s enabled us to identify P450 catalysts for the selective hydroxylation of four aliphatic positions in the target substrates, including a C(sp(3))-H site not oxidized by the parent enzyme. Furthermore, we discovered a general activity-enhancing effect of active-site substitutions involving the unnatural amino acid para-amino-Phe, which resulted in P450 catalysts capable of supporting the highest total turnover number reported to date on a complex molecule (34,650). The functional changes induced by the unnatural amino acids could not be reproduced by any of the 20 natural amino acids. This study thus demonstrates that unnatural amino acid mutagenesis constitutes a promising new strategy for improving the catalytic activity and regioselectivity of P450 oxidation catalysts.
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