Cytochrome P450 engineering

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This article covers protein engineering [1] of cytochrome (CYP) P450 enzymes. P450s are involved in a range of biochemical catabolic and anabolic process. [2] Natural P450s can perform several different types of chemical reactions including hydroxylations, N,O,S-dealkylations, epoxidations, sulfoxidations, aryl-aryl couplings, ring contractions and expansions, oxidative cyclizations, alcohol/aldehyde oxidations, desaturations, nitrogen oxidations, decarboxylations, nitrations, as well as oxidative and reductive dehalogenations. [2] [3] Engineering efforts often strive for 1) improved stability 2) improved activity 3) improved substrate scope 4) enabled ability to catalyze unnatural reactions. [4] [5] P450 engineering is an emerging field in the areas of chemical biology and synthetic organic chemistry (chemoenzymatic).

Contents

Approaches to engineering cytochrome P450 enzymes

Rational

Rational enzyme engineering is characterized by making specific amino acid mutations based on mechanistic or structural information. While P450 enzymes are mechanistically well understood, mutations based on structural information are often limited by crystallization difficulty. [4] [5] Although, when obtainable, the high degree of flexibility and active site plasticity present in P450s make crystal structures largely obsolete for rational design. [5] Another issue presents itself when attempting to expand substrate scope. This is often achieved by increasing the P450 active site size, which in turn can result in multiple substrate docking orientations, resulting in poor regio-/stereoselectivity. [5]

Directed evolution

Directed evolution is an enzyme engineering strategy designed to mimic natural selection in a laboratory setting. [2] [4] [5] Due to the difficulty in implementing rational design strategies, directed evolution has become the strategy of choice for P450 engineering. Here, mutations can be introduced either semi-rationally or randomly via site-saturation mutagenesis. The resulting P450 mutant (typically mutant library) is then screened for desired activity. [5] [6] [7] Mutants displaying enhanced properties are forwarded to subsequent rounds of mutagenesis, repeating this cycle until the desired function is adequately met.

P450 engineering examples

P450 BM3

P450 BM3 (also known as CYP102A1) is a cytochrome P450 enzyme isolated from Bacillus megaterium . [2] [4] [5] [6] [8] BM3 has been extensively studied in the context of enzyme engineering due to its solubility, tractable bacterial isoforms and self-sufficient electron transport system, but also due to its synthetic utility. [8] Engineering studies have revealed that BM3 mutants can 1) be endowed with new and differentiated substrate scopes 2) exhibit regio-/stereoselectivity on new substrates and 3) be engineered to be highly selective and active towards new substrates. [5] [6] [8] BM3 variants have been particularly useful to produce fragrances, flavors, pheromones and pharmaceuticals. [8] Artemisinic acid (used in the production of the pharmaceutical natural product artemisinin) was produced utilizing a BM3 variant responsible for epoxidizing the two alkenes present in amorpha-4,11-diene. [8] [9] Oxidation of valencene to nootkatone (a prized grapefruit flavor) was accomplished utilizing a F87T and I263A mutant (Figure 1). [8]

Figure 1. Oxidation of (+)-valencene utilizing P450BM3 F87T I263A mutant Figure 1. The main oxidation products from the reaction of (+)-valencene with P450cam mutants.png
Figure 1. Oxidation of (+)-valencene utilizing P450BM3 F87T I263A mutant

Recently, Wang et al. reported a BM3 variant capable of performing styrenyl olefin cyclopropanation. [6] As native BM3 displays poor cyclopropanation activity, an enzyme engineering effort was undertaken. At their core, P450s are heme-thiolate enzymes which utilize molecular oxygen (O2) and NAD(P)H to perform oxygenation reactions. [10] As such, BM3 prefers to perform epoxidation opposed to cyclopropanation reactions in the presence of olefins. [6] The reaction between ethyl diazoacetate (EDA) and 1 was chosen as a model reaction due to the known difficulty of epoxidizing electron-deficient olefins utilizing transitional metal catalysis (Figure 2). [6] This reaction generates compound 2, which can easily be converted to levomilnacipran (Fetzima), a pharmaceutical used to treat clinical depression. [6] To begin, mutants were generated where the axial coordinating cysteine residue in the catalytic center was replaced with amino acids serine, alanine, methionine, histidine and tyrosine. The mutant T268A-axH, which has an axial histidine ligand catalyzed the reaction between EDA and 1 in 81% yield with 6:94 diastereoselectivity and 42% enantioselectivity. [6] Subsequent rounds of site-saturation mutagenesis were then performed, resulted in the variant named BM3-Hstar (containing T268A-axH, L437W, V78M and L181V mutations), which could catalyze the model reaction with greater than 92% yield, 92% enantioselectivity and 2:98 diastereoselectivity. [6] As an added advantage, BM3-Hstar was also capable of performing the desired cyclopropanation reaction in the presence of atmospheric oxygen (O2) (the only known BM3 variant capable of this). [6]

Figure 2. Reaction between 1 and ethyl diazoacetate (EDA) catalyzed by BM3-Hstar Figure 2. Reaction between 1 and ethyl diazoacetate (EDA) catalyzed by BM3-Hstar.png
Figure 2. Reaction between 1 and ethyl diazoacetate (EDA) catalyzed by BM3-Hstar

CYP125

Aside from their synthetic utility, P450 enzymes have also been engineered to better understand their biochemistry. [10] Based on the proposed catalytic cycle, an axially ligated thiolate moiety (cysteine) donates electron density to the metal center aiding in protonation of a ferric-peroxo anion intermediate (−O-O-Fe3+) which upon water lose generates a C-H bond reactive iron-oxo species (O=Fe4+). [2] [5] [8] [10] Alternatively, if the ferric-peroxo anion remains un-protonated, this reactive species can mediate C-C bond cleavage in aldehyde-containing substrates (deformylation). [10] In order to better understand intermediate dichotomy between the ferric-peroxo anion and iron-oxo species, CYP125 (which is responsible various metabolic processes including cholesterol degradation) was engineered to replace the axial ligated cysteine residue with selenocysteine (SeCYP125). In turn, it was observed that SeCYP125 favors formation of oxidized products vs deformylated products when reacted with cholesterol-26-aldehyde, indicating that increased electron donation from selenocysteine relative to cysteine results in a higher proportion of iron-oxo relative to ferric-peroxo anion (Figure 3). [10]

Figure 3. Comparison of axial ligand effects on the ferric-peroxo anion in CYP125's catalytic cycle Figure 3. Comparison of axial ligand effects on the ferric-peroxo anion in the P450 catalytic cycle of CYP125.png
Figure 3. Comparison of axial ligand effects on the ferric-peroxo anion in CYP125's catalytic cycle

Ir(Me)-CYP119-Max

In 2016, work published by Dydio et al. reported an artificial metalloenzyme capable of catalyzing intra-/intermolecular carbene C-H insertions into activated/unactivated C-H bonds, with kinetics like that of a native enzyme (Figure 4). The reported catalyst was developed by switching the iron-protoporphyrin cofactor in thermostable P450 enzyme CYP119A1 with an iridium-methyl-protoporphyrin cofactor (Ir(Me)-PIX), followed by directed evolution. CYP119-Max, a quadruple mutant (C317G, T213G, L69V, V254L), was subsequently obtained. Enantiomeric excesses (ee’s) of up to ±98% were obtained with a fixed catalyst loading of 0.17 mol %. CYP119-Max can also undergo intermolecular insertion reactions, albeit with moderate ee (68%). To demonstrate the applicability of CYP119-Max in the production of fine chemicals, a 200 mM scale reaction produced ethyl-2,3-dihydrobenzofuran-3-carboxylate in 44% yield, with 35,000 turnover number (TON) and 93% ee. [7]

Figure 4. Reactions catalyzed by metalloenzyme CYP119 Ir(Me)-PIX-CYP119 mutants Figure 4. Reactions catalyzed by CYP119 mutant Ir(Me)-PIX-CYP119 C317G.png
Figure 4. Reactions catalyzed by metalloenzyme CYP119 Ir(Me)-PIX-CYP119 mutants

Related Research Articles

Active site Active region of an enzyme

In biology, the active site is the region of an enzyme where substrate molecules bind and undergo a chemical reaction. The active site consists of amino acid residues that form temporary bonds with the substrate and residues that catalyse a reaction of that substrate. Although the active site occupies only ~10–20% of the volume of an enzyme, it is the most important part as it directly catalyzes the chemical reaction. It usually consists of three to four amino acids, while other amino acids within the protein are required to maintain the tertiary structure of the enzymes.

Cytochrome P450 Class of enzymes

Cytochromes P450 (CYPs) are a superfamily of enzymes containing heme as a cofactor that functions as monooxygenases. In mammals, these proteins oxidize steroids, fatty acids, and xenobiotics, and are important for the clearance of various compounds, as well as for hormone synthesis and breakdown. In 1963, Estabrook, Cooper, and Rosenthal described the role of CYP as a catalyst in steroid hormone synthesis and drug metabolism. In plants, these proteins are important for the biosynthesis of defensive compounds, fatty acids, and hormones.

CYP3A4 Enzyme which breaks down foreign organic molecules

Cytochrome P450 3A4 is an important enzyme in the body, mainly found in the liver and in the intestine. It oxidizes small foreign organic molecules (xenobiotics), such as toxins or drugs, so that they can be removed from the body. It is highly homologous to CYP3A5, another important CYP3A enzyme.

CYP2E1 Protein-coding gene in the species Homo sapiens

Cytochrome P450 2E1 is a member of the cytochrome P450 mixed-function oxidase system, which is involved in the metabolism of xenobiotics in the body. This class of enzymes is divided up into a number of subcategories, including CYP1, CYP2, and CYP3, which as a group are largely responsible for the breakdown of foreign compounds in mammals.

Drug metabolism is the metabolic breakdown of drugs by living organisms, usually through specialized enzymatic systems. More generally, xenobiotic metabolism is the set of metabolic pathways that modify the chemical structure of xenobiotics, which are compounds foreign to an organism's normal biochemistry, such as any drug or poison. These pathways are a form of biotransformation present in all major groups of organisms and are considered to be of ancient origin. These reactions often act to detoxify poisonous compounds. The study of drug metabolism is called pharmacokinetics.

Any enzyme system that includes cytochrome P450 protein or domain can be called a P450-containing system.

Nitrite reductase refers to any of several classes of enzymes that catalyze the reduction of nitrite. There are two classes of NIR's. A multi haem enzyme reduces NO2− to a variety of products. Copper containing enzymes carry out a single electron transfer to produce nitric oxide.

21-Hydroxylase Human enzyme that hydroxylates steroids

Steroid 21-hydroxylase is an enzyme that hydroxylates steroids at the C21 position and is involved in biosynthesis of aldosterone and cortisol. The enzyme converts progesterone and 17α-hydroxyprogesterone into 11-deoxycorticosterone and 11-deoxycortisol, respectively, within metabolic pathways that ultimately lead to aldosterone and cortisol. Deficiency in the enzyme may cause congenital adrenal hyperplasia.

Steroid 11β-hydroxylase

Steroid 11β-hydroxylase, also known as steroid 11β-monooxygenase, is a steroid hydroxylase found in the zona glomerulosa and zona fasciculata of the adrenal cortex. Named officially the cytochrome P450 11B1, mitochondrial, it is a protein that in humans is encoded by the CYP11B1 gene. The enzyme is involved in the biosynthesis of adrenal corticosteroids by catalyzing the addition of hydroxyl groups during oxidation reactions.

Lanosterol 14 alpha-demethylase

Lanosterol 14α-demethylase (CYP51A1) is the animal version of a cytochrome P450 enzyme that is involved in the conversion of lanosterol to 4,4-dimethylcholesta-8(9),14,24-trien-3β-ol. The cytochrome P450 isoenzymes are a conserved group of proteins that serve as key players in the metabolism of organic substances and the biosynthesis of important steroids, lipids, and vitamins in eukaryotes. As a member of this family, lanosterol 14α-demethylase is responsible for an essential step in the biosynthesis of sterols. In particular, this protein catalyzes the removal of the C-14α-methyl group from lanosterol. This demethylation step is regarded as the initial checkpoint in the transformation of lanosterol to other sterols that are widely used within the cell.

In chemistry, bis(oxazoline) ligands (often abbreviated BOX ligands) are a class of privileged chiral ligands containing two oxazoline rings. They are typically C2‑symmetric and exist in a wide variety of forms; with structures based around CH2 or pyridine linkers being particularly common (often generalised BOX and PyBOX respectively). The coordination complexes of bis(oxazoline) ligands are used in asymmetric catalysis. These ligands are examples of C2-symmetric ligands.

In enzymology, a secologanin synthase (EC 1.14.19.62, was wrongly classified as EC 1.3.3.9 in the past) is an enzyme that catalyzes the chemical reaction

In enzymology, an ethylbenzene hydroxylase (EC 1.17.99.2) is an enzyme that catalyzes the chemical reaction

Thiosulfate dehydrogenase

Thiosulfate dehydrogenase is an enzyme that catalyzes the chemical reaction:

Organogold chemistry is the study of compounds containing gold–carbon bonds. They are studied in academic research, but have not received widespread use otherwise. The dominant oxidation states for organogold compounds are I with coordination number 2 and a linear molecular geometry and III with CN = 4 and a square planar molecular geometry. The first organogold compound discovered was gold(I) carbide Au2C2, which was first prepared in 1900.

A transition metal oxo complex is a coordination complex containing an oxo ligand. Formally O2-, an oxo ligand can be bound to one or more metal centers, i.e. it can exist as a terminal or (most commonly) as bridging ligands (Fig. 1). Oxo ligands stabilize high oxidation states of a metal. They are also found in several metalloproteins, for example in molybdenum cofactors and in many iron-containing enzymes. One of the earliest synthetic compounds to incorporate an oxo ligand is potassium ferrate (K2FeO4), which was likely prepared by Georg E. Stahl in 1702.

Steroid 15beta-monooxygenase (EC 1.14.15.8, cytochrome P-450meg, cytochrome P450meg, steroid 15beta-hydroxylase, CYP106A2, BmCYP106A2) is an enzyme with systematic name progesterone,reduced-ferredoxin:oxygen oxidoreductase (15beta-hydroxylating) . This enzyme catalyses the following chemical reaction

Galactose oxidase

Galactose oxidase is an enzyme that catalyzes the oxidation of D-galactose in some species of fungi.

An artificial metalloenzyme (ArM) is a metalloprotein made in the laboratory which cannot be found in the nature and can catalyze certain desired chemical reactions. Despite fitting into classical enzyme categories, ArMs also have potential in chemical reactivity like catalyzing Suzuki coupling, metathesis and so on, which are never reported in natural enzymatic reaction. With the progress in organometallic synthesis and protein engineering, more and more new kind of design of ArMs came out, showing promising future in both academia and industrial aspects.

Cytochrome P450 aromatic O-demethylase

Cytochrome P450 aromatic O-demethylase is a bacterial enzyme that catalyzes the demethylation of lignin and various lignols. The net reaction follows the following stoichiometry, illustrated with a generic methoxy arene:

References

  1. ↑ Brannigan, James; Wilkinson, Anthony (December 2002). "Protein engineering 20 years on". Nature Reviews Molecular Cell Biology. 3 (12): 964–70. doi:10.1038/nrm975. PMID   12461562.
  2. 1 2 3 4 5 McIntosh, John; Farwell, Christopher; Arnold, Frances (March 20, 2014). "Expanding P450 catalytic reaction space through evolution and engineering". Current Opinion in Chemical Biology. 19: 126–134. doi:10.1016/j.cbpa.2014.02.001. PMC   4008644 . PMID   24658056.
  3. ↑ Munro, Andrew; Girvan, Hazel; McLean, Kirsty (December 15, 2006). "Variations on a (t)heme-novel mechanisms, redox partners and catalytic functions in the cytochrome P450 superfamily". Natural Product Reports. 24 (3): 585–609. doi:10.1039/B604190F. PMID   17534532.
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  5. 1 2 3 4 5 6 7 8 9 Fasan, Rudi (February 22, 2012). "Tuning P450 Enzymes as Oxidation Catalysts". ACS Catalysis. 2 (4): 647–666. doi:10.1021/cs300001x. S2CID   4674699.
  6. 1 2 3 4 5 6 7 8 9 10 Wang, Z. Jane; Renata, Hans; Peck, Nicole; Farwell, Christopher; Coelho, Pedro; Arnold, Frances (May 5, 2014). "Improved Cyclopropanation Activity of Histidine-Ligated Cytochrome P450 Enables the Enantioselective Formal Synthesis of Levomilncipran". Angewandte Chemie International Edition in English. 53 (26): 6810–6813. doi:10.1002/anie.201402809. PMC   4120663 . PMID   24802161.
  7. 1 2 Dydio, P.; Key, H.; Nazarenko, A.; Rha, J.; Seyedkazemi, V.; Clark, D.; Hartwig, John (October 7, 2016). "An artificial metalloenzyme with the kinetics of native enzymes". Science. 354 (6308): 102–106. Bibcode:2016Sci...354..102D. doi: 10.1126/science.aah4427 . PMID   27846500.
  8. 1 2 3 4 5 6 7 Whitehouse, Christopher; Bell, Stephen; Wong, Luet-Lok (July 18, 2012). "P450BM3 (CYP102A1): connecting the dots". Chemical Society Reviews. 41 (3): 1218–1260. doi:10.1039/C1CS15192D. PMID   22008827.
  9. ↑ van Agtmael, Michiel; Eggelte, Teunis; van Boxtel, Chris (May 1, 1999). "Artemisinin drugs in the treatment of malaria: from medicinal herb to registered medication". Trends in Pharmacological Sciences. 20 (5): 199–205. doi:10.1016/S0165-6147(99)01302-4. PMID   10354615.
  10. 1 2 3 4 5 Sivaramakrishnan, Santhosh; Ouellet, Hugues; Matsumura, Hirotoshi; Guan, Shenheng; Loccoz, Pierre; Burlingame, Alma; Montellano, Paul (March 23, 2012). "Proximal Ligand Electron Donation and Reactivity of the Cytochrome P450 Ferric-Peroxo Anion". Journal of the American Chemical Society. 134 (15): 6673–6684. doi:10.1021/ja211499q. PMC   3329582 . PMID   22444582.
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