Suicide inactivation of peroxidases and the challenge of engineering more robust enzymes

As the number of industrial applications for proteins continues to expand, the exploitation of protein engineering becomes critical. It is predicted that protein engineering can generate enzymes with new catalytic properties and create desirable, high-value, products at lower production costs. Peroxidases are ubiquitous enzymes that catalyze a variety of oxygen-transfer reactions and are thus potentially useful for industrial and biomedical applications. However, peroxidases are unstable and are readily inactivated by their substrate, hydrogen peroxide. Researchers rely on the powerful tools of molecular biology to improve the stability of these enzymes, either by protecting residues sensitive to oxidation or by devising more efficient intramolecular pathways for free-radical allocation. Here, we discuss the catalytic cycle of peroxidases and the mechanism of the suicide inactivation process to establish a broad knowledge base for future rational protein engineering.     

A Mechanistic Understanding of the Distinct Regio- and Chemoselectivity of Multifunctional P450s by Structural Comparison of IkaD and CftA Complexed with Common Substrates

Regio- and chemoselective C−H activation at multi-positions of a single molecule is fascinating but chemically challenging. The homologous cytochrome P450 enzymes IkaD and CftA catalyze multiple C−H oxidations on the same polycyclic tetramate macrolactam (PoTeM) ikarugamycin, with distinct regio- and chemoselectivity. Herein we provide mechanistic understanding of their functional differences by solving crystal structures of IkaD and CftA in complex with ikarugamycin and unnatural substrates. Distinct conformations of the F/G region in IkaD and CftA are found to differentiate the orientation of PoTeM substrates, by causing different binding patterns with polar moieties to determine site selection, oxidation order, and chemoselectivity. Fine-tuning the polar subpocket altered the regioselectivity of IkaD, indicating that substrate re-orientation by mutating residues distal to the oxidation site could serve as an important method in future engineering of P450 enzymes.     

Selective Enzymatic Oxidation of Silanes to Silanols

Compared to the biological world's rich chemistry for functionalizing carbon, enzymatic transformations of the heavier homologue silicon are rare. We report that a wild-type cytochrome P450 monooxygenase (P450_BM3 from Bacillus megaterium, CYP102A1) has promiscuous activity for oxidation of hydrosilanes to give silanols. Directed evolution was applied to enhance this non-native activity and create a highly efficient catalyst for selective silane oxidation under mild conditions with oxygen as the terminal oxidant. The evolved enzyme leaves C−H bonds present in the silane substrates untouched, and this biotransformation does not lead to disiloxane formation, a common problem in silanol syntheses. Computational studies reveal that catalysis proceeds through hydrogen atom abstraction followed by radical rebound, as observed in the native C−H hydroxylation mechanism of the P450 enzyme. This enzymatic silane oxidation extends nature's impressive catalytic repertoire.     

Substrate binding to cytochromes P450

P450s have attracted tremendous attention owing to not only their involvement in the metabolism of drug molecules and endogenous substrates but also the unusual nature of the reaction they catalyze, namely, the oxidation of unactivated C–H bonds. The binding of substrates to P450s, which is usually viewed as the first step in the catalytic cycle, has been studied extensively via a variety of biochemical and biophysical approaches. These studies were directed towards answering different questions related to P450s, including mechanism of oxidation, substrate properties, unusual substrate oxidation kinetics, function, and active-site features. Some of the substrate binding studies extending over a period of more than 40 years of dedicated work have been summarized in this review and categorized by the techniques employed in the binding studies.

高通量筛选策略在细胞色素P450单加氧酶定向进化中的应用

细胞色素P450单加氧酶具有催化活性混杂性的特点,可以催化多种氧化反应,因而在生物催化领域受到了极大的关注。然而P450单加氧酶往往存在催化活性低、稳定性差、区域和立体选择性不理想等问题,从而限制了其在生物催化领域的广泛运用。蛋白质定向进化的发展与运用为改善P450单加氧酶的催化性能提供了有效的途径,而一种高效的高通量筛选策略是保证酶蛋白定向进化成功实施的关键。本文综述了P450单加氧酶定向进化过程中高通量筛选策略的最新进展。     

Cytochromes P450 as useful biocatalysts: addressing the limitations

Cytochrome P450 monooxygenases (P450s or CYPs) are a unique family of enzymes which are capable of catalysing the regio- and stereospecific oxidation of non-functionalised hydrocarbons. Despite the enormous synthetic potential of P450s, these enzymes have yet to be extensively employed for research purposes or in industry. Lack of stability, low activity, narrow substrate specificity, expensive cofactor requirements, limited solvent tolerance and electron supply are some of the main reasons why the academic and industrial implementation of these important biocatalysts remains a challenge. Considering the significance of P450s, many research groups have focused on improving their properties in an effort to make more robust catalysts with broad synthetic applications. This article focuses on some of the factors that have limited the exploitation of P450s and explores some of the significant steps that have been taken towards addressing these limitations.     

Diversity of P450 enzymes in the biosynthesis of natural products

Covering: 1985 to 2012Diverse oxygenation patterns of natural products generated by secondary metabolic pathways in microorganisms and plants are largely achieved through the tailoring reactions catalysed by cytochrome P450 enzymes (P450s). P450s are a large family of oxidative hemoproteins found in all life forms from prokaryotes to humans. Understanding the reactivity and selectivity of these fascinating C-H bond-activating catalysts will advance their use in generating valuable pharmaceuticals and products for medicine, agriculture and industry. A major strength of this P450 group is its set of established enzyme-substrate relationships, the source of the most detailed knowledge on how P450 enzymes work. Engineering microbial-derived P450 enzymes to accommodate alternative substrates and add new functions continues to be an important near- and long-term practical goal driving the structural characterization of these molecules. Understanding the natural evolution of P450 structure-function should accelerate metabolic engineering and directed evolutionary approaches to enhance diversification of natural product structures and other biosynthetic applications.     

Synthetic Aspects of Metal-Catalyzed Oxidations of Amines and Related Reactions

The metabolism of amines is governed by a variety of enzymes such as amine oxidase, flavoenzyme, and cytochrome P-450. A wide variety of compounds are produced such as ammonia and alkaloids in selective and clean oxidation reactions that proceed under mild reaction conditions. Simulation of the functions of these enzymes with simple transition metal complex catalysts may lead to the discovery of biomimetic, catalytic oxidations of amines and related compounds. Indeed, metal complex catalyzed oxidations have been found to proceed with high efficiency. The first section of this review discusses the dehydrogenative oxidations of amines with transition metal catalysts by transition metal catalysts that simulate amine oxidase. The second section highlights the catalytic oxidation of secondary amines to nitrones by simulation of flavoenzymes. The third section describes the simulation of the function of cytochrome P-450 with lowvalent ruthenium complexes and peroxides. Biomimetic ruthenium-catalyzed oxidations of tertiary amines, secondary amines, and other substrates such as amides, β-lactams, nitriles, alcohols, alkenes, ketones, and even nonactivated hydrocarbons can be performed selectively under mild conditions. These three general approaches provide highly useful strategies for synthesis of fine chemicals and biologically active compounds such as alkaloids, amino acids, and β-lactams.     

Association of Cytochrome P450 Enzymes is a Determining Factor in their Catalytic Activity

Previously, our laboratory demonstrated that one cytochrome P450 isoenzyme can influence the catalytic properties of another P450 isoenzyme when combined in a reconstituted system. Moreover, our data and that of other investigators indicate that P450 interaction is required for catalytic activity even when one isoenzyme is present. The goal of the current study was to examine the possible mechanism of these interactions in more detail. Analyzing recently published X-ray data of microsomal P450 enzymes and protein docking studies, four types of dimer formations of P450 enzymes were examined in more detail. In case of two dimer types, the aggregating partner was shown to contribute to NADPH cytochrome P450 reductase (CPR) binding-a flavoprotein whose interaction with P450 is required for expressing P450 functional activity of the neighboring P450 moiety. Thus, it was shown that dimerization of P450 enzymes might result in an altered affinity towards the CPR. Two dimer types were shown to exist only in the presence of a substrate, while the other two types exist also without a substrate present. The molecular basis was established for the fact that the presence of a substrate and other P450 enzymes simultaneously determine the catalytic activity. Furthermore, a kinetic model was improved describing the catalytic activity of P450 enzymes as a function of CPR concentration based on equilibrium between different supramolecular organizations of P450 enzymes. This model was successfully applied in order to explain our experimental data and that of other investigators.Eszter Hazai and Zsolt Bikádi contributed equally to this workDavid Kupfer-Deceased     

Fluorescence detection of ligand binding to labeled cytochrome P450 BM3

The cytochrome P450 superfamily of monoxygenases are highly relevant for pharmaceutical, environmental and biocatalytical applications. The binding of a substrate to their catalytic site is usually detectable by UV-vis spectroscopy as a low-to-high spin state transition of the heme iron. However, the discovery of potential new substrates is limited by the fact that some compounds do not cause the typical spin-shift even if they are oxidised by P450 enzymes. Here we report a fluorescence-based method able to detect the binding of such substrates to the heme domain of cytochrome P450 BM3 from Bacillus megaterium. The protein was labeled with the fluorescent probe N,N'-dimethyl-N-(iodoacetyl)-N'-(7-nitrobenz-2-oxa-1,3-diazol-4-Yl)-ethylenediamine (IANBD). Arachidonic and lauric acids are substrates of P450 BM3 and were used to validate the method, as their binding can be detected both by a spin-shift of the Soret peak from 419 to 397 nm and by the fluorescence change of the labelled protein. The fluorescence emission of the probe linked to the protein increased by a value corresponding to 121 ± 9% and 52 ± 5% with respect to the initial one, upon titration with arachidonic or lauric acids respectively. The dissociation constants were calculated by both UV-vis and fluorescence spectroscopy. Three drugs, propranolol, chlorzoxazone and nifedipine, known to be oxidized by P450 BM3 and that bind without causing spin-shift, were also tested and the fluorescence emission of IANBD was found to decrease by 29 ± 5%, 21 ± 2% and 23 ± 3%, respectively, allowing the measurement of their dissociation constants.     

Significance of Protein–Substrate Hydrogen Bonding for the Selectivity of P450-Catalysed Oxidations

P450_cin and P450_cam are bacterial cytochromes P450 that specifically hydroxylate bicyclic monoterpenes. Protein–substrate H bonding has been previously proposed as crucial in the selectivity of P450_cin oxidations, but not as essential for P450_cam. To examine the difference in importance of H bonds in these two model P450s, the P450-catalysed oxidation products from thiocamphor were compared. Surprisingly, both P450s oxidised thiocamphor predominantly to the corresponding S-oxides, in contrast to previous reports, and this is the first report of P450-catalysed sulfine generation from a thioketone. Additionally, the result emphasised the importance of the protein–substrate H bond to selectivity in both P450_cin and P450_cam. The H bonding in P450_cam was re-examined using camphane, another substrate for which the protein–substrate H bond is absent. The results indicated that both H bonding and hydrophobic interactions between substrate and protein play a role in selectivity. Interestingly, the protein–substrate H bond was not a factor in substrate affinity for the enzyme.     

Theoretical studies and industrial applications of oxidative activation of inert C-H bond by metalloporphyrin-based biomimetic catalysis

High costs and low catalytic efficiency of metalloporphyrins, which are an analogue of cytochrome P450 enzyme, are the bot-tlenecks in the industrialization of biomimetic hydrocarbon oxidation reactions. The basic principle and research technique of physical organic chemistry were applied to the process of biomimetic oxidation of hydrocarbon catalyzed by metalloporphyrins. This biomimetic technology could be adapted to bulk chemicals production by developing the new methods for efficient scale-up synthesis of metalloporphyrins, new pathways for molecular oxygen activation on an industrial scale and new approaches to elevate the catalytic efficiency of metalloporphyrins. This review mainly focuses on research carried out in our group.     

Engineering and assaying of cytochrome P450 biocatalysts

Cytochrome P450s constitute a highly fascinating superfamily of enzymes which catalyze a broad range of reactions. They are essential for drug metabolism and promise industrial applications in biotechnology and biosensing. The constant search for cytochrome P450 enzymes with enhanced catalytic performances has generated a large body of research. This review will concentrate on two key aspects related to the identification and improvement of cytochrome P450 biocatalysts, namely the engineering and assaying of these enzymes. To this end, recent advances in cytochrome P450 development are reported and commonly used screening methods are surveyed.     

P450(BM3) (CYP102A1): connecting the dots

P450(BM3) (CYP102A1), a fatty acid hydroxylase from Bacillus megaterium, has been extensively studied over a period of almost forty years. The enzyme has been redesigned to catalyse the oxidation of non-natural substrates as diverse as pharmaceuticals, terpenes and gaseous alkanes using a variety of engineering strategies. Crystal structures have provided a basis for several of the catalytic effects brought about by mutagenesis, while changes to reduction potentials, inter-domain electron transfer rates and catalytic parameters have yielded functional insights. Areas of active research interest include drug metabolite production, the development of process-scale techniques, unravelling general mechanistic aspects of P450 chemistry, methane oxidation, and improving selectivity control to allow the synthesis of fine chemicals. This review draws together the disparate research themes and places them in a historical context with the aim of creating a resource that can be used as a gateway to the field.     

Metabolism of Halogenated Alkanes by Cytochrome P450 enzymes. Aerobic Oxidation versus Anaerobic Reduction

The cytochromes P450 are a large class of heme‐containing enzymes that catalyze a broad range of chemical reactions in biosystems, mainly through oxygen‐atom transfer to substrates. A relatively unknown reaction catalyzed by the P450s, but very important for human health, is the activation of halogenated substrates, which may lead to toxicity problems. However, its catalytic mechanism is currently unknown and, therefore, we performed a detailed computational study. To gain insight into the metabolism of halogenated compounds by P450 enzymes, we have investigated the oxidative and reductive P450‐mediated activation of tetra‐ and trichloromethane as halogenated models with density functional theory (DFT) methods. We propose an oxidative halosylation mechanism for CCl₄ under aerobic conditions by Compound I of P450, which follows the typical Groves‐type rebound mechanism. By contrast, the metabolism of CHCl₃ occurs preferentially via an initial hydrogen‐atom abstraction rather than halosylation. Kinetic isotope effect studies should, therefore, be able to distinguish the mechanistic pathways of CCl₄ versus CHCl₃. We find a novel mechanism that is different from the well accepted P450 substrate activation mechanisms reported previously. Moreover, the studies highlight the substrate specific activation pathways by P450 enzymes leading to different products. These reactivity differences are rationalized using Marcus theory equations, which reproduce experimental product distributions.     

Is the bound substrate in nitric oxide synthase protonated or neutral and what is the active oxidant that performs substrate hydroxylation?

We present here results of a series of density functional theory (DFT) studies on enzyme active site models of nitric oxide synthase (NOS) and address the key steps in the catalytic cycle whereby the substrate (L-arginine) is hydroxylated to N(omega)-hydroxo-arginine. It has been proposed that the mechanism follows a cytochrome P450-type catalytic cycle; however, our calculations find an alternative low energy pathway whereby the bound L-arginine substrate has two important functions in the catalytic cycle, namely first as a proton donor and later as the substrate in the reaction mechanism. Thus, the DFT studies show that the oxo-iron active species (compound I) cannot abstract a proton and neither a hydrogen atom from protonated L-arginine due to the strength of the N-H bonds of the substrate. However, the hydroxylation of neutral arginine by compound I and its one electron reduced form (compound II) requires much lower barriers and is highly exothermic. Detailed analysis of proton transfer mechanisms shows that the basicity of the dioxo dianion and the hydroperoxo-iron (compound 0) intermediates in the catalytic cycle are larger than that of arginine, which makes it likely that protonated arginine donates one of the two protons needed during the first catalytic cycle of NOS. Therefore, DFT predicts that in NOS enzymes arginine binds to the active site in its protonated form, but is deprotonated during the oxygen activation process in the catalytic cycle by either the dioxo dianion species or compound 0. As a result of the low ionization potential of neutral arginine, the actual hydroxylation reaction starts with an initial electron transfer from the substrate to compound I to create compound II followed by a concerted hydrogen abstraction/radical rebound from the substrate. These studies indicate that compound II is the actual oxidant in NOS enzymes that performs the hydroxylation reaction of arginine, which is in sharp contrast with the cytochromes P450 where compound II was shown to be a sluggish oxidant. This is the first example of an enzyme where compound II is able to participate in the reaction mechanism. Moreover, arginine hydroxylation by NOS enzymes is catalyzed in a significantly different way from the cytochromes P450 although the active sites of the two enzyme classes are very similar in structure. Detailed studies of environmental effects on the reaction mechanism show that environmental perturbations as appear in the protein have little effect and do not change the energies of the reaction. Finally, a valence bond curve crossing model has been set up to explain the obtained reaction mechanisms for the hydrogen abstraction processes in P450 and NOS enzymes.     

How do azoles inhibit cytochrome P450 enzymes? A density functional study

To examine how azole inhibitors interact with the heme active site of the cytochrome P450 enzymes, we have performed a series of density functional theory studies on azole binding. These are the first density functional studies on azole interactions with a heme center and give fundamental insight into how azoles inhibit the catalytic function of P450 enzymes. Since azoles come in many varieties, we tested three typical azole motifs representing a broad range of azole and azole-type inhibitors: methylimidazolate, methyltriazolate, and pyridine. These structural motifs represent typical azoles, such as econazole, fluconazole, and metyrapone. The calculations show that azole binding is a stepwise mechanism whereby first the water molecule from the resting state of P450 is released from the sixth binding site of the heme to create a pentacoordinated active site followed by coordination of the azole nitrogen to the heme iron. This process leads to the breaking of a hydrogen bond between the resting state water molecule and the approaching inhibitor molecule. Although, formally, the water molecule is released in the first step of the reaction mechanism and a pentacoordinated heme is created, this does not lead to an observed spin state crossing. Thus, we show that release of a water molecule from the resting state of P450 enzymes to create a pentacoordinated heme will lead to a doublet to quartet spin state crossing at an Fe-OH(2) distance of approximately 3.0 A, while the azole substitution process takes place at shorter distances. Azoles bind heme with significantly stronger binding energies than a water molecule, so that these inhibitors block the catalytic cycle of the enzyme and prevent oxygen binding and the catalysis of substrate oxidation. Perturbations within the active site (e.g., a polarized environment) have little effect on the relative energies of azole binding. Studies with an extra hydrogen-bonded ethanol molecule in the model, mimicking the active site of the CYP121 P450, show that the resting state and azole binding structures are close in energy, which may lead to chemical equilibrium between the two structures, as indeed observed with recent protein structural studies that have demonstrated two distinct azole binding mechanisms to P450 heme.     

Bioelectrodes for evaluating molecular therapeutic and toxicity properties

Recent progress on material designs merged with nanotechnology and biotechnology strategies has advanced studies of complex biological samples on electrodes for cytochrome P450 (CYP)–driven biocatalytic reactions (e.g. liver membrane fractions, cells, and various organ-specific CYP extracts). In addition, protein engineering of CYP enzymes with their reductase partner in membranes (e.g. baculovirus- or Escherichia coli bacteria–expressed CYP microsomes) and other recombinant strategies (e.g. engineered CYP and reductase fusion domains and site-directed CYP mutagenesis) are promising sustainable approaches for offering abundant sources of CYP enzymes for electrocatalytic applications. The combination of in silico and experimental electroanalytical methods with hyphenated approaches and biological assays can offer early and rigorous profiling of new drugs and specialty chemicals for safe exposure and beneficial use.     

Selective Oxidations Using a Cytochrome P450 Enzyme Variant Driven with Surrogate Oxygen Donors and Light

Cytochrome P450 monooxygenase enzymes are versatile catalysts, which have been adapted for multiple applications in chemical synthesis. Mutation of a highly conserved active site threonine to a glutamate can convert these enzymes into peroxygenases that utilise hydrogen peroxide (H₂O₂). Here, we use the T252E-CYP199A4 variant to study peroxide-driven oxidation activity by using H₂O₂ and urea-hydrogen peroxide (UHP). We demonstrate that the T252E variant has a higher stability to H₂O₂ in the presence of substrate that can undergo carbon-hydrogen abstraction. This peroxygenase variant could efficiently catalyse O-demethylation and an enantioselective epoxidation reaction (94 % ee). Neither the monooxygenase nor peroxygenase pathways of the P450 demonstrated a significant kinetic isotope effect (KIE) for the oxidation of deuterated substrates. These new peroxygenase variants offer the possibility of simpler cytochrome P450 systems for selective oxidations. To demonstrate this, a light driven H₂O₂ generating system was used to support efficient product formation with this peroxygenase enzyme.     

Evolutionary relationship and application of a superfamily of cyclic amidohydrolase enzymes

Cyclic amidohydrolases belong to a superfamily of enzymes that catalyze the hydrolysis of cyclic C-N bonds. They are commonly found in nucleotide metabolism of purine and pyrimidine. These enzymes share similar catalytic mechanisms and show considerable structural homologies, suggesting that they might have evolved from a common ancestral protein. Homology searches based on common mechanistic properties and three-dimensional protein structures provide clues to the evolutionary relationships of these enzymes. Among the superfamily of enzymes, hydantoinase has been highlighted by its potential for biotechnological applications in the production of unnatural amino acids. The enzymatic process for the production of optically pure amino acids consists of three enzyme steps: hydantoin racemase, hydantoinase, and N-carbamoylase. For efficient industrial application, some critical catalytic properties such as thermostability, catalytic activity, enantioselectivity, and substrate specificity require further improvement. To this end, isolation of new enzymes with desirable properties from natural sources and the optimization of enzymatic processes were attempted. A combination of directed evolution techniques and rational design approaches has made brilliant progress in the redesign of industrially important catalytic enzymes; this approach is likely to be widely applied to the creation of designer enzymes with desirable catalytic properties.