LICRED: A Versatile Drop‐In Vector for Rapid Generation of Redox‐Self‐Sufficient Cytochrome P450s

Cytochromes P450 (P450s) are a family of haem‐containing oxidases with considerable potential as tools for industrial biocatalysis. Organismal genomes are revealing thousands of gene sequences that encode P450s of as yet unknown function, the exploitation of which will require high‐throughput tools for their isolation and characterisation. In this report, a ligationindependent cloning vector “LICRED” is described that enables the high‐throughput generation of libraries of redox‐self‐sufficient P450s by fusing a range of P450 haem domains to the reductase of P450RhF (RhF‐Red) in a robust and generically applicable way. Cloning and expression of fusions of RhF‐Red with the haem domains of P450cam and P450‐XplA resulted in soluble, active, redox‐self‐sufficient, chimeric enzymes. In vitro studies also revealed that electron transfer from NADPH to haem was primarily intramolecular. The general applicability of the LICRED platform was then demonstrated through the creation of a library of RhF‐Red fusion constructs by using the diverse complement of P450 haem domains identified in the genome of Nocardia farcinica. The resultant fusion‐protein library was then screened against a panel of substrates; this revealed chimeric enzymes competent for the hydroxylation of testosterone and methyltestosterone, and the dealkylation of 7‐ethoxycoumarin.     

Harnessing P450 Enzyme for Biotechnology and Synthetic Biology

Cytochrome P450 enzymes (P450s, CYPs) catalyze the oxidative transformation of a wide range of organic substrates. Their functions are crucial to xenobiotic metabolism and steroid transformation in humans and other organisms. The enzymes are promising for synthetic biology applications but limited by several drawbacks including low turnover rates, poor stability, the dependance of expensive cofactors and redox partners, and the narrow substrate scope. To conquer these obstacles, emerging strategies including substrate engineering, usage of decoy and decoy-based small molecules auxiliaries, designing of artificial enzyme cascades and the incorporation of materials have been explored based on the unique properties of P450s. These strategies can be applied to a wide range of P450s and can be combined with protein engineering to improve the enzymatic activities. This minireview will focus on some recent developments of these strategies which have been used to leverage P450 catalysis. Remaining challenges and future opportunities will also be discussed.     

A Gene‐Fusion Approach to Enabling Plant Cytochromes P450 for Biocatalysis

Cytochromes P450 from plants have the potential to be valuable catalysts for industrial hydroxylation reactions, but their application is hindered by poor solubility, the lack of suitable expression systems and the requirement of P450s for auxiliary redox‐transport proteins for the delivery of reducing equivalents from NAD(P)H. In the interests of enabling useful P450 activity from plants, we have developed a suite of vectors for the expression of plant P450s as non‐natural genetic fusions with reductase proteins. First, we have fused the P450 isoflavone synthase (IFS) from Glycine max with the bacterial P450 reductase domain (Rhf‐RED) from Rhodococcus sp., by using our LICRED vector developed previously (F. Sabbadin, R. Hyde, A. Robin, E.‐M. Hilgarth, M. Delenne, S. Flitsch, N. Turner, G. Grogan, N. C. Bruce, ChemBioChem 2010 , 11, 987–994) creating the first active bacterial–plant fusion P450 enzyme. We have then created a complementary vector, ACRyLIC for the fusion of selected plant P450 enzymes to the P450 reductase ATR2 from Arabidopsis thaliana. The applicability of this vector to the creation of active P450 fusion enzymes was demonstrated using both IFS1 and the cinnamate‐4‐hydroxylase (C4H) from A. thaliana. Overall the fusion vector systems will allow the rapid creation of libraries of plant P450s with the aim of identifying enzyme activities with possible applications in industrial biocatalysis.     

Molecular Evolution of a Steroid Hydroxylating Cytochrome P450 Using a Versatile Steroid Detection System for Screening

Molecular evolution is a powerful tool for improving or changing activities of enzymes for their use in biotechnological processes. Cytochromes P450 are highly interesting enzymes for biotechnological purposes because they are able to hydroxylate a broad variety of substrates with high regio- and stereoselectivity. One promising steroid hydroxylating cytochrome P450 for biotechnological applications is CYP106A2 from Bacillus megaterium ATCC 13368. It is one of a few known bacterial cytochromes P450 able to transform steroids such as progesterone and 11-deoxycortisol. CYP106A2 can be easily expressed in Escherichia coli with a high yield and can be reconstituted using the adrenal redox proteins, adrenodoxin and adrenodoxin reductase. We developed a simple screening assay for this system and performed random mutagenesis of CYP106A2, yielding variants with improved 11-deoxycortisol and progesterone hydroxylation activity. After two generations of directed evolution, we were able to improve the k (cat)/K (m) of the 11-deoxycortisol hydroxylation by a factor of more than four. At the same time progesterone conversion was improved about 1.4-fold. Mapping the mutations identified in catalytically improved CYP106A2 variants into the structure of a CYP106A2 model suggests that these mutations influence the mobility of the F/G loop, and the interaction with the redox partner adrenodoxin. The results show the evolution of a soluble steroid hydroxylase as a potential new catalyst for the production of steroidogenic compounds.     

Cluster Screening: An Effective Approach for Probing the Substrate Space of Uncharacterized Cytochrome P450s

Cytochrome P450 monooxygenases (P450s) are versatile enzymes with high potential for biocatalysis. The number of newly annotated P450 genes has been increasing constantly, and these thus represent a rich resource for new biocatalysts. However, the substrate scopes of newly identified P450s are often not known, and thus their exploitation is difficult. Herein we describe an approach, named “cluster screening”, and its application for the primary characterization of two P450s: CYP154E1 and CYP154A8. A library comprising 51 compounds was designed and organized into nine groups according to their chemical properties. The activities of both P450s in vitro were maintained with suitable nonphysiological redox partners, and the cluster library was screened with these enzymes for product formation. From this library, 30 compounds tested positive for CYP154E1 and 23 were positive for CYP154A8. Cluster screening distinguishes subtle differences in activity and selectivity of enzymes as closely related as those of the same P450 family. For example, the alkaloid pergolide mesylate was converted by CYP154E1 (4 %) but not by CYP154A8. A building block of vitamin D₃, Grundmann's ketone, was converted by both enzymes, although conversion was higher with CYP154E1 (100 vs 53 %).     

Bringing out the Potential of Wild-type Cytochrome P450s using Decoy Molecules: Oxygenation of Nonnative Substrates by Bacterial Cytochrome P450s

To bring out the potential of wild-type cytochrome P450s, we have developed a series of “decoy molecules” to change their high substrate specificity without any mutagenesis. Decoy molecules are inert dummy substrates with structures that are very similar to those of natural substrates. The decoy molecules force long-alkyl-chain fatty acid hydroxylases (P450_BSβ, P450_SPα, and P450BM3) to generate the active species and to catalyze oxidation of various substrates other than fatty acids. Interestingly, the catalytic activity was highly dependent on the structure of decoy molecules. Furthermore, the enantioselectivity of reactions catalyzed by P450_BSβ and P450_SPα was also dependent on the structure of decoy molecules. The decoy molecule system allows us to control reactions catalyzed by wild-type enzymes by designing decoy molecules.     

Conformational Landscape of Cytochrome P450 Reductase Interactions

Oxidative reactions catalyzed by Cytochrome P450 enzymes (CYPs), which constitute the most relevant group of drug-metabolizing enzymes, are enabled by their redox partner Cytochrome P450 reductase (CPR). Both proteins are anchored to the membrane of the endoplasmic reticulum and the CPR undergoes a conformational change in order to interact with the respective CYP and transfer electrons. Here, we conducted over 22 microseconds of molecular dynamics (MD) simulations in combination with protein–protein docking to investigate the conformational changes necessary for the formation of the CPR–CYP complex. While some structural features of the CPR and the CPR–CYP2D6 complex that we highlighted confirmed previous observations, our simulations revealed additional mechanisms for the conformational transition of the CPR. Unbiased simulations exposed a movement of the whole protein relative to the membrane, potentially to facilitate interactions with its diverse set of redox partners. Further, we present a structural mechanism for the susceptibility of the CPR to different redox states based on the flip of a glycine residue disrupting the local interaction network that maintains inter-domain proximity. Simulations of the CPR–CYP2D6 complex pointed toward an additional interaction surface of the FAD domain and the proximal side of CYP2D6. Altogether, this study provides novel structural insight into the mechanism of CPR–CYP interactions and underlying conformational changes, improving our understanding of this complex machinery relevant for drug metabolism.     

What controls the rates of interprotein electron-transfer reactions

Rates of electron-transfer (ET) reactions are dependent on driving force, reorganizational energy, distance, and the nature of the medium which the electron must traverse. In kinetically complex biological systems, non-ET reactions may be required to activate the system for ET and may also influence the observed rates. Studies of ET from tryptophan tryptophylquinone to copper to heme in the methylamine dehydrogenase-amicyanin-cytochrome c-551i ET complex, as well as studies of other physiologic redox protein complexes, are used to illustrate the combination of factors which control rates of interprotein ET reactions.     

Sesquiterpenoids Produced by Combining Two Sesquiterpene Cyclases with Promiscuous Myxobacterial CYP260B1

Sesquiterpenes represent a class of important terpenoids with high structural diversity and a wide range of applications. The cyclized core skeletons are generated by sesquiterpene cyclases, and the structural diversity is further increased by a series of modification steps. Cytochromes P450 (P450s) are a class of monooxygenases and one of the main contributors to the structural diversity of natural products. Some of these P450s show a broad substrate range and might be promising candidates for the implementation of cascade reactions. In this study, a combinatorial biosynthesis approach was utilized by the combination of a promiscuous myxobacterial P450 (CYP260B1) with two sesquiterpene cyclases (FgJ01056, FgJ09920) of filamentous fungi. Two oxygenated products, culmorin and culmorone, and a new compound, koraidiol, were successfully generated and characterized. This approach suggests the potential use of noncognate P450s to produce novel oxygenated terpenoids, or to generate a novel biosynthetic route for known terpenoids by a combinatorial biosynthesis strategy.     

Product Distributions of Cytochrome P450 OleTJE with Phenyl-Substituted Fatty Acids: A Computational Study

There are two types of cytochrome P450 enzymes in nature, namely, the monooxygenases and the peroxygenases. Both enzyme classes participate in substrate biodegradation or biosynthesis reactions in nature, but the P450 monooxygenases use dioxygen, while the peroxygenases take H₂O₂ in their catalytic cycle instead. By contrast to the P450 monooxygenases, the P450 peroxygenases do not require an external redox partner to deliver electrons during the catalytic cycle, and also no external proton source is needed. Therefore, they are fully self-sufficient, which affords them opportunities in biotechnological applications. One specific P450 peroxygenase, namely, P450 OleT_JE, reacts with long-chain linear fatty acids through oxidative decarboxylation to form hydrocarbons and, as such, has been implicated as a suitable source for the biosynthesis of biofuels. Unfortunately, the reactions were shown to produce a considerable amount of side products originating from C^α and C^β hydroxylation and desaturation. These product distributions were found to be strongly dependent on whether the substrate had substituents on the C^α and/or C^β atoms. To understand the bifurcation pathways of substrate activation by P450 OleT_JE leading to decarboxylation, C^α hydroxylation, C^β hydroxylation and C^α–C^β desaturation, we performed a computational study using 3-phenylpropionate and 2-phenylbutyrate as substrates. We set up large cluster models containing the heme, the substrate and the key features of the substrate binding pocket and calculated (using density functional theory) the pathways leading to the four possible products. This work predicts that the two substrates will react with different reaction rates due to accessibility differences of the substrates to the active oxidant, and, as a consequence, these two substrates will also generate different products. This work explains how the substrate binding pocket of P450 OleT_JE guides a reaction to a chemoselectivity.     

Intramolecular electron transfer in a cytochrome P450cam system with a site-specific branched structure

Cytochrome P450 (P450) is an attractive oxygenase due to the diverse catalytic reactions and the broad substrate specificity. Class I P450s require an excess concentration (more than 10 times) of iron-sulfur proteins, which transfer electrons to P450s, to attain the maximum catalytic activity and this requirement is a critical bottleneck for practical applications. Here, we show a site-specific branched fusion protein of P450 with its electron transfer proteins using enzymatic cross-linking with transglutaminase. A branched fusion protein of P450 from Pseudomonas putida (P450cam), which was composed of one molecule each of P450cam, putidaredoxin (Pdx) and Pdx reductase, showed higher catalytic activity (306 min(-1)) and coupling efficiency (99%) than the equimolar reconstitution system due to the intramolecular electron transfer. The unique site-specific branched structure simply increased local concentration of proteins without denaturation of each protein. Therefore, enzymatic post-translational protein manipulation can be a powerful alternative to conventional strategies for the creation of multicomponent enzyme systems with novel proteinaceous architecture.     

Amide Neighbouring‐Group Effects in Peptides: Phenylalanine as Relay Amino Acid in Long‐Distance Electron Transfer

In nature, proteins serve as media for long‐distance electron transfer (ET) to carry out redox reactions in distant compartments. This ET occurs either by a single‐step superexchange or through a multi‐step charge hopping process, which uses side chains of amino acids as stepping stones. In this study we demonstrate that Phe can act as a relay amino acid for long‐distance electron hole transfer through peptides. The considerably increased susceptibility of the aromatic ring to oxidation is caused by the lone pairs of neighbouring amide carbonyl groups, which stabilise the Phe radical cation. This neighbouring‐amide‐group effect helps improve understanding of the mechanism of extracellular electron transfer through conductive protein filaments (pili) of anaerobic bacteria during mineral respiration.     

生物燃料电池酶电极的研究进展

综述了生物燃料电池酶电极的研究进展,尤其是近年来在氧化还原酶的种类、电子介体电极、直接电子传递电极以及固定化酶等方面的研究成果。从提高生物燃料电池的转换效率出发,分析各因素对酶电极性能的影响,包括针对不同底物燃料使用相应的氧化还原酶实现电极之间的电子传递、小分子或聚合物中介体存在下提高电流密度、导电聚合物等修饰电极对直接电子传递效率的贡献,以及物理或化学的酶固定化方法增加酶的稳定性等。因此采用新材料及新工艺构筑酶电极,最大程度上保持酶的活性,提高载酶量及电子传递效率,将成为该领域未来的发展方向。     

Challenges in capturing oxygenase activity in vitro

Biocatalysis using oxygenase or desaturase enzymes has the potential to add value to native fats and oils by adding oxygen, hydroxyl groups, or double bonds to create regio- and/or stereospecific products. These enzymes are a subset of the large class of oxidoreductase enzymes (from EC subgroups 1.13 and 1.14) involved with biological oxidation and reduction. In vitro biocatalytic processing using these enzymes is hampered by the high cost of the stoichiometric cofactors. This article reviews recent progress in developing in vitro redox enzyme biocatalysis for commercial-scale syntheses. Coenzyme recycling and electrochemical redox cycling as methods for cofactor regeneration are described and commercial applications indicated. Direct charge transfer without use of mediators is described as the cleanest way of introducing the reducing power into the catalytic cycle. Our electrochemically driven cytochrome P450_cam bioreactor is discussed as an example of direct charge transfer to a redox protein. Site-directed mutagenesis in the active site of the P450_cam monooxygenase greatly improved performance for the conversion of the nonnative substrate, styrene to styrene oxide. This epoxidation reaction was also shown to give a single product (styrene oxide) in the bioelectrochemical reactor when the diatomic oxygen co-substrate was managed properly. Certain commercial equipment, instruments, and materials are identified in this paper to specify adequately the experimental procedure. In no case does such identification imply recommendation or endorsement by the National Institute of Standards and Technology nor does it imply that the material or equipment is necessarily the best available for the purpose.     

Enhanced enzyme activity through electron transfer between single-walled carbon nanotubes and horseradish peroxidase

Better understanding of electron transfer (ET) taking place at the nano-bio interface can guide design of more effective functional materials used in fuel cells, biosensors, and medical devices. Single-walled carbon nanotube (SWCNT) coupled with biological enzymes serves as a model system for studying the ET mechanism, as demonstrated in the present study. SWCNT enhanced the activity of horseradish peroxidase (HRP) in the solution-based redox reaction by binding to HRP at a site proximate to the enzyme's activity center and participating in the ET process. ET to and from SWCNT was clearly observable using near-infrared spectroscopy. The capability of SWCNT in receiving electrons and the direct attachment of HRP to the surface of SWCNT strongly affected the enzyme activity due to the direct involvement of SWCNT in ET.     

Electron Transfer Pathways in Cholesterol Synthesis

Cholesterol synthesis in the endoplasmic reticulum requires electron input at multiple steps and utilizes both NADH and NADPH as the electron source. Four enzymes catalyzing five steps in the pathway require electron input: squalene monooxygenase, lanosterol demethylase, sterol 4α‐methyl oxidase, and sterol C5‐desaturase. The electron‐donor proteins for these enzymes include cytochrome P450 reductase and the cytochrome b₅ pathway. Here I review the evidence for electron donor protein requirements with these enzymes, the evidence for additional electron donor pathways, and the effect of deletion of these redox enzymes on cholesterol and lipid metabolism.     

手性NAD类似物合成及其辅酶应用

烟酰胺腺嘌呤二核苷酸（NAD）是生命体中必不可少的氧化还原辅因子。NAD具有β-NAD和α-NAD两种差向异构体,而大多数NAD依赖酶偏好β-NAD。近年来,已合成大量结构简化的NAD类似物,并探索其作为氧化还原辅因子应用于生物催化反应。通常认为酶天然具有立体选择性,而很多结构简化的NAD类似物不含手性中心,暗示有必要合成手性NAD类似物以评估其性能。本研究拟设计合成手性NAD类似物,并初步探讨部分氧化还原酶利用它们作为辅酶的催化性能。以商品化光学纯的氨基-3-苯基丙酸为手性原料,合成了两对NAD类似物对映体,并用来源于巨芽孢杆菌的突变型细胞色素P450单加氧酶（P450 BM3 R966D/W1046S）和来源于硫化叶菌的突变型葡萄糖脱氢酶（SsGDH I192T/V306I）,测试这些NAD类似物作为辅酶的催化活性。结果发现,P450 BM3 R966D/W1046S利用还原型S构型NAD类似物的催化效率高于对应的R构型NAD类似物;而SsGDH I192T/V306I则对NAD类似物对映体均没有活性。研究初步表明,氨基酸可作为手性原料制备立体构型明确的NAD类似物,而氧化还原酶表现出偏好特定手性NAD类似物的现象,也提示需要合成更多的手性NAD类似物,并系统测试和匹配氧化还原酶及其突变体,以创建出基于辅酶依赖型氧化还原酶的新催化体系。     

Relationship between redox activity and chemical speciation of size-fractionated particulate matter

Background  

Although the mechanisms of airborne particulate matter (PM) related health effects remain incompletely understood, one emerging hypothesis is that these adverse effects derive from oxidative stress, initiated by the formation of reactive oxygen species (ROS) within affected cells. Typically, ROS are formed in cells through the reduction of oxygen by biological reducing agents, with the catalytic assistance of electron transfer enzymes and redox active chemical species such as redox active organic chemicals and metals. The purpose of this study was to relate the electron transfer ability, or redox activity, of the PM samples to their content in polycyclic aromatic hydrocarbons and various inorganic species. The redox activity of the samples has been shown to correlate with the induction of the stress protein, hemeoxygenase-1.     

Structural and electron paramagnetic resonance (EPR) studies of mononuclear molybdenum enzymes from sulfate-reducing bacteria

Molybdenum and tungsten are found in biological systems in a mononuclear form in the active site of a diverse group of enzymes that generally catalyze oxygen-atom-transfer reactions. The metal atom (Mo or W) is coordinated to one or two pyranopterin molecules and to a variable number of ligands such as oxygen (oxo, hydroxo, water, serine, aspartic acid), sulfur (cysteines), and selenium (selenocysteines) atoms. In addition, these proteins contain redox cofactors such as iron-sulfur clusters and heme groups. All of these metal cofactors are along an electron-transfer pathway that mediates the electron exchange between substrate and an external electron acceptor (for oxidative reactions) or donor (for reductive reactions). We describe in this Account a combination of structural and electronic paramagnetic resonance studies that were used to reveal distinct aspects of these enzymes.