Faculty of 1000 evaluation for New reactions and products resulting from alternative interactions between the P450 enzyme and redox partners (original) (raw)
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Scientific Reports, 2017
Most bacterial cytochrome P450 monooxygenases (P450s or CYPs) require two redox partner proteins for activity. To reduce complexity of the redox chain, the Bacillus subtilis flavodoxin YkuN (Y) was fused to the Escherichia coli flavodoxin reductase Fpr (R), and activity was tuned by placing flexible (GGGGS) n or rigid ([E/L]PPPP) n linkers (n = 1-5) in between. P-linker constructs typically outperformed their G-linker counterparts, with superior performance of YR-P5, which carries linker ([E/L]PPPP) 5. Molecular dynamics simulations demonstrated that ([E/L]PPPP) n linkers are intrinsically rigid, whereas (GGGGS) n linkers are highly flexible and biochemical experiments suggest a higher degree of separation between the fusion partners in case of long rigid P-linkers. The catalytic properties of the individual redox partners were best preserved in the YR-P5 construct. In comparison to the separate redox partners, YR-P5 exhibited attenuated rates of NADPH oxidation and heme iron (III) reduction, while coupling efficiency was improved (28% vs. 49% coupling with B. subtilis CYP109B1, and 44% vs. 50% with Thermobifida fusca CYP154E1). In addition, YR-P5 supported monooxygenase activity of the CYP106A2 from Bacillus megaterium and bovine CYP21A2. The versatile YR-P5 may serve as a non-physiological electron transfer system for exploitation of the catalytic potential of other P450s. Cytochrome P450 monooxygenases (CYPs or P450s) are highly versatile heme containing enzymes that catalyse a wide variety of oxidation reactions, while accepting a large diversity of substrates. The impressive range of reactions catalysed by P450s includes amongst others hydroxylation, epoxidation, dealkylation and deamination, as well as unusual reactions such as aromatic dehalogenation and Baeyer-Villiger oxidation 1-3. Illustrative of their diverse biological functions, P450s are capable of converting amongst others fatty acids, steroids, prostaglandins, terpenes, and xenobiotics such as drugs and antibiotics 4, 5. It is therefore not surprising that these multipurpose biocatalysts have become attractive targets for application in biotechnology and synthetic biology 6. P450s catalyse the reductive scission of molecular oxygen upon which one oxygen atom is introduced into the non-activated substrate, while the second atom is reduced to water 7. Activation of molecular oxygen relies on the successive delivery of two electrons derived from the pyridine cofactor NAD(P)H to the heme iron, which is typically facilitated by a dedicated redox partner system 8, 9. Based on the number of proteins involved, two major types of P450 redox systems can be distinguished: (i) two-component systems consisting of an FAD-containing reductase and either an iron-sulfur containing ferredoxin or an FMN-containing flavodoxin. These systems are commonly found in bacteria and mitochondria, and (ii) a single diflavin (FAD/FMN) P450 reductase (CPR) that supports the function of eukaryotic microsomal P450s. A limited number of bacterial P450s have been identified that carry such a CPR-like module as a fused functional domain (for example CYP102A1 from Bacillus subtilis) 8, 9 .
Journal of Biological Chemistry, 2012
Background: A hierarchy of catalytic steps characterizes multifunctional cytochrome P450 enzymes. Results: In the post-polyketide oxidative tailoring of mycinamicins by MycG, the two methoxy groups of mycinose are sensors that mediate initial recognition and discriminate between closely related molecules. Conclusion: Bulky and conformationally restrained macrolide substrates advance to the catalytically productive mode through multiple steps. Significance: Protein engineering facilitating substrate progression may enhance catalysis.
Redox Partners: Function Modulators of Bacterial P450 Enzymes
Trends in Microbiology, 2020
The superfamily of cytochrome P450 monooxygenases (P450s) is widespread in all kingdoms of life. Functionally versatile P450s are extensively involved in diverse anabolic and catabolic processes. P450s require electrons to be transferred by redox partners (RPs) for O 2 activation and substrate monooxygenation. Unlike monotonic eukaryotic cytochrome P450 reductases, bacterial RP systems are more diverse and complicated. Recent studies have demonstrated that the type, the amount, the combination, and the mode of action of bacterial RPs can affect not only the catalytic rate and product distribution but also the type and selectivity of P450 reactions. These results are leading to a novel opinion that RPs not only function as auxiliary electron transfer proteins but are also important P450 function modulators. Cytochrome P450 Monooxygenases Highlights Ubiquitous P450s catalyze various oxidative reactions towards an enormous number of substrates. Bacterial P450s in soluble forms represent the most diverse subset with great application value and potential.
Biotechnology and Applied Biochemistry, 2013
This review covers the current state of knowledge regarding artificial fusion constructs of cytochrome P450 enzymes in which the activity of the catalytic heme is driven by reductases of different origins. Cytochromes P450 form a vast family of heme-thiolate proteins, which act as monooxygenases by activating molecular oxygen, resulting in the insertion of one atom into an organic substrate with the concomitant reduction of the other to water. The reducing equivalents are usually supplied by nicotinamide adenine dinucleotide or nicotinamide adenine dinucleotide phosphate and are transferred in two consecutive steps via the redox partner(s). These include reductases containing flavin mononucleotide and/or flavin adenine dinucleotide and/or Fe-S clusters in different combinations depending on the P450 system. These enzymes catalyze extremely diverse reactions, including regioand stereospecific oxidations of a large range of substrates in addition to many drugs and xenobiotics, as well as biosynthesis of physiologically important compounds such as various steroids, vitamins, and lipids. Because of their ability to catalyze such a vast range of reactions, they have become the focus of biotechnological interest, but their dependence on the reductase partner has remained one of the challenging limitations for full exploration of their synthetic potential. To address the latter limitation, many researchers have reconstituted functional P450 enzymes by fusion with different reductase proteins; this review will cover their findings.
Advances in Experimental Medicine and Biology, 2015
Cytochrome P450 enzymes (P450s) have the ability to oxidize unactivated C-H bonds of substrates with remarkable regio-and stereoselectivity. Comparable selectivity for chemical oxidizing agents is typically difficult to achieve. Hence, there is an interest in exploiting P450s as potential biocatalysts. Despite their impressive attributes, the current use of P450s as biocatalysts is limited. While bacterial P450 enzymes typically show higher activity, they tend to be highly selective for one or a few substrates. On the other hand, mammalian P450s, especially the drugmetabolizing enzymes, display astonishing substrate promiscuity. However, product prediction continues to be challenging. This review discusses the use of small molecules for controlling P450 substrate specificity and product selectivity. The focus will be on two approaches in the area: (1) the use of decoy molecules, and (2) the application of substrate engineering to control oxidation by the enzyme.
Bioorganic & Medicinal Chemistry, 2014
P450 enzymes (P450s) are well known for their ability to oxidize unactivated C-H bonds with high regio-and stereoselectivity. Hence, there is emerging interest in exploiting P450s as potential biocatalysts. Although bacterial P450s typically show higher activity than their mammalian counterparts, they tend to be more substrate selective. Most drug-metabolizing P450s on the other hand, display remarkable substrate promiscuity, yet product prediction remains challenging. Protein engineering is one established strategy to overcome these issues. A less explored, yet promising alternative involves substrate engineering. This review discusses the use of small molecules for controlling the substrate specificity and product selectivity of P450s. The focus is on two approaches, one taking advantage of non-covalent decoy molecules, and the other involving covalent substrate modifications.2009 Elsevier Ltd. All rights reserved.
An A245T Mutation Conveys on Cytochrome P450eryF the Ability to Oxidize Alternative Substrates
Journal of Biological Chemistry, 2000
Cytochrome P450 eryF (CYP107A1), which hydroxylates deoxyerythronolide B in erythromycin biosynthesis, lacks the otherwise highly conserved threonine that is thought to promote O-O bond scission. The role of this threonine is satisfied in P450 eryF by a substrate hydroxyl group, making deoxyerythronolide B the only acceptable substrate. As shown here, replacement of Ala 245 by a threonine enables the oxidation of alternative substrates using either H 2 O 2 or O 2 /spinach ferredoxin/ferredoxin reductase as the source of oxidizing equivalents. Testosterone is oxidized to 1-, 11␣-, 12-, and 16␣-hydroxytestosterone. A kinetic solvent isotope effect of 2.2 indicates that the A245T mutation facilitates dioxygen bond cleavage. This gain-of-function evidence confirms the role of the conserved threonine in P450 catalysis. Furthermore, a Hill coefficient of 1.3 and dependence of the product distribution on the testosterone concentration suggest that two testosterone molecules bind in the active site, in accord with a published structure of the P450 eryF-androstenedione complex. P450 eryF is thus a structurally defined model for the catalytic turnover of multiply bound substrates proposed to occur with CYP3A4. In view of its large active site and defined structure, catalytically active P450 eryF mutants are also attractive templates for the engineering of novel P450 activities.
Applications of microbial cytochrome P450 enzymes in biotechnology and synthetic biology
Current opinion in chemical biology, 2016
Cytochrome P450 enzymes (P450s) are a superfamily of monooxygenase enzymes with enormous potential for synthetic biology applications. Across Nature, their substrate range is vast and exceeds that of other enzymes. The range of different chemical transformations performed by P450s is also substantial, and continues to expand through interrogation of the properties of novel P450s and by protein engineering studies. The ability of P450s to introduce oxygen atoms at specific positions on complex molecules makes these enzymes particularly valuable for applications in synthetic biology. This review focuses on the enzymatic properties and reaction mechanisms of P450 enzymes, and on recent studies that highlight their broad applications in the production of oxychemicals. For selected soluble bacterial P450s (notably the high-activity P450-cytochrome P450 reductase enzyme P450 BM3), variants with a multitude of diverse substrate selectivities have been generated both rationally and by rando...
Scientific Reports, 2019
Information about substrate and product selectivity is critical for understanding the function of cytochrome P450 monooxygenases. In addition, comprehensive understanding of changes in substrate selectivity of P450 upon amino acid mutation would enable the design and creation of engineered P450s with desired selectivities. Therefore, systematic methods for obtaining such information are required. Herein, we developed an integrated P450 substrate screening system for the selection of “exemplary” substrates for a P450 of interest. The established screening system accurately selected the known exemplary substrates and also identified previously unknown exemplary substrates for microbial-derived P450s from a library containing sp3-rich synthetic small molecules. Synthetically potent transformations were also found by analyzing the reactions and oxidation products. The screening system was applied to analyze the substrate selectivity of the P450 BM3 mutants F87A and F87A/A330W, which acq...
Design of a Novel P450: A Functional Bacterial−Human Cytochrome P450 Chimera †
Biochemistry, 1998
We report the construction of a functional chimera from approximately 50% bacterial (cytosolic) cytochrome P450cam and 50% mammalian (membrane-bound) cytochrome P450 2C9. The chimeric protein shows a reduced CO-difference spectrum absorption at 446 nm, and circular dichroism spectra indicate that the protein is globular. The protein is soluble and catalyzes the oxidation of 4-chlorotoluene using molecular oxygen and reducing equivalents from bacterial putidaredoxin and putidaredoxin reductase. This chimera provides a novel method for addressing structure-function issues and may prove useful in the design of oxidants for benign and stereospecific synthesis, as well as catalysts for bioremediation of polluted areas. Furthermore, these results provide the first evidence that bacterial P450 enzymes and mammalian P450 enzymes are likely to share a common tertiary structure.