Replacement of Natural Cofactors by Selected Hydrogen Peroxide Donors or Organic Peroxides Results in Improved Activity for CYP3A4 and CYP2D6

  title={Replacement of Natural Cofactors by Selected Hydrogen Peroxide Donors or Organic Peroxides Results in Improved Activity for CYP3A4 and CYP2D6},
  author={Amandine Chefson and Jin Zhao and Karine Auclair},
The cytochrome P450 enzymes (P450s or CYPs) form a ubiquitous family of heme proteins able to catalyze the monooxygenation of a wide range of substrates. P450s are of considerable interest in synthetic organic chemistry because of their impressive ability to catalyze the insertion of oxygen into nonactivated C H bonds. This useful reaction in organic chemistry has received much attention over several decades, but still remains a significant challenge. Some metal catalysts have been successfully… 
Directed evolution of cytochrome P450 enzymes for biocatalysis: exploiting the catalytic versatility of enzymes with relaxed substrate specificity.
Progress that has been made in altering properties of P450s such as substrate range, cofactor preference and stability is illustrated, and some of the remaining challenges that must be overcome for industrial application of these powerful biocatalysts are outlined.
Monooxygenase, peroxidase and peroxygenase properties and reaction mechanisms of cytochrome P450 enzymes.
It has been difficult to isolate the historically elusive CpdI intermediate in the native NAD(P)H/O2-supported monooxygenase pathway and to determine its precise electronic structure and kinetic and physicochemical properties because of its high reactivity, unstable nature and short life cycle.
An Inexpensive, Efficient Alternative to NADPH to Support Catalysis by Thermostable Cytochrome P450 Enzymes
Using two candidate P450s, it is shown that OS‐dependent P450 catalysis can be optimized in a few steps, boosting product yield from ∼2.2 % with CPR to 88–100‬% with an OS.
Progress towards the easier use of P450 enzymes.
The attempts to overcome limitations of biocatalysts using approaches such as mutagenesis, chemical modifications, conditions engineering and immobilization are covered.
Catalysis with Cytochrome P450 Monooxygenases
This chapter focuses on the recent progress in applying P450 monooxygenases as biocatalysts and the P450 engineering issue will also be discussed.
Mechanistic basis of electron transfer to cytochromes p450 by natural redox partners and artificial donor constructs.
  • P. Hlavica
  • Biology, Chemistry
    Advances in experimental medicine and biology
  • 2015
Deeper insight into the mechanistic basis of the redox machinery will permit optimization of redox processes via directed evolution and DNA shuffling and help obviate the tedious reconstitution procedure and induces novel activities.
The activity of human CYP2D6 in low water organic solvents
It is reported here that CYP2D6 colyophilized with trehalose and suspended in n‐decane shows higher activity than in aqueous buffer, and provides an alternative strategy to facilitate the use of this enzyme in synthesis.
Use of chemical auxiliaries to control p450 enzymes for predictable oxidations at unactivated C-h bonds of substrates.
The use of small molecules for controlling P450 substrate specificity and product selectivity is discussed, including the use of decoy molecules and the application of substrate engineering to control oxidation by the enzyme.
Monooxygenase, Peroxidase and Peroxygenase Properties and Mechanisms of Cytochrome P450
This work discusses Cytochrome P450 Enzymes and Electrochemistry: Crosstalk with Electrodes as Redox Partners and Electron Sources, Mechanistic Basis of Electron Transfer to Cytochromes P450 by Natural Redox partners and Artificial Donor Constructs, and Biological Diversity of Cyto chrome P450 Redox Partner Proteins.
Water Oxidation by a Cytochrome P450: Mechanism and Function of the Reaction
This reaction has an adaptive value to bacteria that express this camphor catabolism pathway, which requires O2, for two reasons: 1) the borneol and H2O2 mixture generated is toxic to other bacteria and 2) borneol down-regulates the expression of P450cam and its electron transfer partners.


Laboratory evolution of peroxide-mediated cytochrome P450 hydroxylation
The directed evolution of the P450 from Pseudomonas putida is reported to create mutants that hydroxylate naphthalene in the absence of cofactors through the ‘peroxide shunt’ pathway, with more than 20-fold higher activity than the native enzyme.
A self-sufficient peroxide-driven hydroxylation biocatalyst.
A self-sufficient P450 BM-3 variant which utilizes hydrogen peroxide (H2O2) to catalyze hydroxylation and epoxidation at high rates and offers an opportunity to employ cellfree P450 catalysis without requiring NAD(P)H regeneration, additional proteins, or dioxygen, and eliminates rate-limiting electron-transfer steps.
Regioselectivity and Activity of Cytochrome P450 BM‐3 and Mutant F87A in Reactions Driven by Hydrogen Peroxide
While significantly enhancing peroxygenase activity, the F87A mutation also shifts hydroxylation further away from the terminal position, and the H2O2- driven reactions with either the full-length BM-3 enzyme or the heme domain are slow, but yield product distributions very similar to those generated when using NADPH and O2.
Engineering Cytochrome P450 BM-3 for Oxidation of Polycyclic Aromatic Hydrocarbons
The high activities of the mutant towards polycyclic aromatic hydrocarbons indicate the potential of engineering P450 BM-3 for the biodegradation of these compounds in the environment.
Identification of the heme-modified peptides from cumene hydroperoxide-inactivated cytochrome P450 3A4.
The incremental masses detected by electrospray mass spectrometric analyses of the heme-modified peptides are consistent with a dipyrrolic heme fragment comprised of either pyrrole ring A-D or B-C, a known soluble product of peroxidative heme degradation, as a modifying species.