A new cytochrome P450 system from Bacillus megaterium DSM319 for the hydroxylation of 11-keto-β-boswellic acid (KBA)

In the genome of Bacillus megaterium DSM319, a strain who has recently been sequenced to fully exploit its potential for biotechnological purposes, we identified a gene encoding the cytochrome P450 CYP106A1 as well as genes encoding potential redox partners of CYP106A1. We cloned, expressed, and purified CYP106A1 and five potential autologous redox partners, one flavodoxin and four ferredoxins. The flavodoxin and three ferredoxins were able to support the activity of CYP106A1 displaying the first cloned natural redox partners of a cytochrome P450 from B. megaterium. The CYP106A1 system was able to convert the pentacyclic triterpene 11-keto-β-boswellic acid (KBA) belonging to the main bioactive constituents of Boswellia serrata gum resin extracts, which are used to treat inflammatory disorders and arthritic diseases. In order to provide sufficient amounts of the KBA products to characterize them structurally by NMR spectroscopy, recombinant whole-cell biocatalysts were constructed based on B. megaterium MS941. The main product has been identified as 7β-hydroxy-KBA, while the side product (～20 %) was shown to be a mixture of 7β,15α-dihydroxy-KBA and 15α-hydroxy-KBA. Without further optimization 560.7 mg l^?1 day^?1 of the main product, 7β-hydroxy-KBA, could be obtained thus providing a suitable starting point for the efficient production of modified KBA by chemical tailoring to produce novel KBA derivatives with increased bioavailability and this way more efficient drugs.     

CYP109E1 is a novel versatile statin and terpene oxidase from Bacillus megaterium

CYP109E1 is a cytochrome P450 monooxygenase from Bacillus megaterium with a hydroxylation activity for testosterone and vitamin D3. This study reports the screening of a focused library of statins, terpene-derived and steroidal compounds to explore the substrate spectrum of this enzyme. Catalytic activity of CYP109E1 towards the statin drug-precursor compactin and the prodrugs lovastatin and simvastatin as well as biotechnologically relevant terpene compounds including ionones, nootkatone, isolongifolen-9-one, damascones, and β-damascenone was found in vitro. The novel substrates induced a type I spin-shift upon binding to P450 and thus permitted to determine dissociation constants. For the identification of conversion products by NMR spectroscopy, a B. megaterium whole-cell system was applied. NMR analysis revealed for the first time the ability of CYP109E1 to catalyze an industrially highly important reaction, the production of pravastatin from compactin, as well as regioselective oxidations generating drug metabolites (6′β-hydroxy-lovastatin, 3′α-hydroxy-simvastatin, and 4″-hydroxy-simvastatin) and valuable terpene derivatives (3-hydroxy-α-ionone, 4-hydroxy-β-ionone, 11,12-epoxy-nootkatone, 4(R)-hydroxy-isolongifolen-9-one, 3-hydroxy-α-damascone, 4-hydroxy-β-damascone, and 3,4-epoxy-β-damascone). Besides that, a novel compound, 2-hydroxy-β-damascenone, produced by CYP109E1 was identified. Docking calculations using the crystal structure of CYP109E1 rationalized the experimentally observed regioselective hydroxylation and identified important amino acid residues for statin and terpene binding.

     

CYP106A2—A versatile biocatalyst with high potential for biotechnological production of selectively hydroxylated steroid and terpenoid compounds

CYP106A2 from Bacillus megaterium ATCC13368, was identified in the 1970s as one of the first bacterial steroid hydroxylases responsible for the conversion of progesterone to 15β-hydroxyprogesterone. Later on it has been proven to be a potent hydroxylase of numerous 3-oxo-Δ⁴ as well as 3-hydroxy-Δ⁵-steroids and has recently also been characterized as a regioselective allylic bacterial diterpene hydroxylase. The main hydroxylation position of CYP106A2 is thought to be influenced by the functional groups at C3 position in the steroid core leading to a favored 15β-hydroxylation of 3-oxo-Δ⁴-steroids and 7β-hydroxylation of 3-hydroxy-Δ⁵-steroids. However, in some cases the hydroxylation is not strictly selective, resulting in the formation of undesired side-products. To overcome the unspecific hydroxylations or, on the contrary, to gain more of these products in case they are of industrial interest, rational protein design and directed evolution have been successfully performed to shift the stereoselectivity of hydroxylation by CYP106A2. The subsequently obtained hydroxylated steroid and terpene derivatives are especially useful as drug metabolites and drug precursors for the pharmaceutical industry, due to their diverse biological properties and hardship of their chemical synthesis. As a soluble prokaryotic P450 with broad substrate spectrum and hydroxylating capacity, CYP106A2 is an outstanding candidate to establish bioconversion processes. It has been expressed with respectable yields in Escherichia coli and Bacillus megaterium and was applied for the preparative hydroxylation of several steroids and terpenes. Recently, the application of the enzyme was assessed under process conditions as well, depicting a successfully optimized process development and getting us closer to industrial scale process requirements and a future large scale application. This article is part of a Special Issue entitled: Cytochrome P450 biodiversity and biotechnology, edited by Erika Plettner, Gianfranco Gilardi, Luet Wong, Vlada Urlacher, Jared Goldstone.     

A new <Emphasis Type="Italic">Bacillus megaterium</Emphasis> whole-cell catalyst for the hydroxylation of the pentacyclic triterpene 11-keto-β-boswellic acid (KBA) based on a recombinant cytochrome P450 system

The use of cytochromes P450 for the regio- and stereoselective hydroxylation of non-activated carbon atoms in biotechnological applications reflects an efficient and cost-effective alternative in comparison to classical organic chemistry. The prokaryotic cytochrome P450 CYP106A2 from Bacillus megaterium ATCC 13368 hydroxylates a variety of 3-oxo-Δ⁴ steroids and recently it was identified to carry out a one-step regioselective allylic hydroxylation of the diterpene abietic acid. The anti-inflammatory pentacyclic triterpene 11-Keto-β-boswellic acid (KBA) was found to be a further substrate of CYP106A2, being the first report of a pentacyclic triterpene conversion by a prokaryotic P450. The reaction products were analyzed by HPLC and the corresponding kinetic parameters were investigated. Structure determination of the main product by NMR revealed a 15α-hydroxylation of this substrate. In order to overcome the inability of a recombinant P450 whole-cell system in E. coli for the uptake of acids with terpene structure, we developed for the first time an expression system for cytochromes P450 in B. megaterium (strains MS941 and ATCC 13368). Interestingly, CYP106A2 was only successfully expressed in the plasmid-less B. megaterium strain MS941 but not in ATCC13368. This recombinant system, with the co-expressed heterologous redox chain of the P450, bovine adrenodoxin reductase (AdR), and bovine adrenodoxin (Adx), was applied for the whole-cell conversion of KBA. The formation of 15α-hydroxy-KBA was increased 15-fold in comparison with the naturally CYP106A2-expressing B. megaterium strain ATCC 13368.     

Identification of monohydroxy progesterones produced by CYP106A2 using comparative HPLC and electrospray ionisation collision-induced dissociation mass spectrometry

Two previously uncharacterised products, produced by recombinant CYP106A2 of Bacillus megaterium ATCC 13368 using progesterone as substrate, were identified. For this purpose a combination of comparative HPLC and electrospray ionisation collision induced dissociation mass spectrometry (ESI CID MS) was established and applied for rapid identification of the steroids, which were identified as 11alpha-hydroxyprogesterone and 9alpha-hydroxyprogesterone. The pharmaceutical relevance of these steroids is discussed. Furthermore, the hydroxylation activity was quantified for all monohydroxylation products (15beta-hydroxyprogesterone, 6beta-hydroxyprogesterone, 11alpha-hydroxyprogesterone, and 9alpha-hydroxyprogesterone). The V(max) values for 15beta-hydroxyprogesterone, 6beta-hydroxyprogesterone, 11alpha-hydroxyprogesterone, and 9alpha-hydroxyprogesterone were determined as 337.3+/-43.7, 22.3+/-0.9, 17.5+/-0.9, and 6.5+/-0.3nmol product/min/nmol CYP106A2, respectively.     

Microbial production of two new dihydroxylated androstenedione derivatives by Acremonium strictum

We found that Acremonium strictum NN106 converted 4-androstene-3, 17-dione (androstenedione) to 12 compounds. Among them, five products were isolated and found to be hydroxylated at the 11α-, 14α-, 7α, 11α-, 6β, 11α- or 6β, 14α-positions of androstenedione. 6β, 11α-Dihydroxy and 6β, 14α-dihydroxy derivatives of androstenedione have been obtained for the first time. From the time course profile of this transformation, sequential hydroxylation at the 6β-position followed by 11α- or 14α-monohydroxylation was observed. The oxidative product of the 6β, 14α-dihydroxy derivative was found to be the most potent inhibitor of human placentral aromatase.     

CYP264B1 from Sorangium cellulosum So ce56: a fascinating norisoprenoid and sesquiterpene hydroxylase

Many terpenes and terpenoid compounds are known as bioactive substances with desirable fragrance and medicinal activities. Modification of such compounds to yield new derivatives with desired properties is particularly attractive. Cytochrome P450 monooxygenases are potential enzymes for these reactions due to their capability of performing different reactions on a variety of substrates. We report here the characterization of CYP264B1 from Sorangium cellulosum So ce56 as a novel sesquiterpene hydroxylase. CYP264B1 was able to convert various sesquiterpenes including nootkatone and norisoprenoids (α-ionone and β-ionone). Nootkatone, an important grapefruit aromatic sesquiterpenoid, was hydroxylated mainly at position C-13. The product has been shown to have the highest antiproliferative activity compared with other nootkatone derivatives. In addition, CYP264B1 was found to hydroxylate α- and β-ionone, important aroma compounds of floral scents, regioselectively at position C-3. The products, 3-hydroxy-β-ionone and 13-hydroxy-nootkatone, were confirmed by (1)H and (13)C NMR. The kinetics of the product formation was analyzed by high-performance liquid chromatography, and the K ( m ) and k (cat) values were calculated. The results of docking α-/β-ionone and nootkatone into a homology model of CYP264B1 revealed insights into the structural basis of these selective hydroxylations.     

Rice CYP734As function as multisubstrate and multifunctional enzymes in brassinosteroid catabolism

Catabolism of brassinosteroids regulates the endogenous level of bioactive brassinosteroids. In Arabidopsis thaliana, bioactive brassinosteroids such as castasterone (CS) and brassinolide (BL) are inactivated mainly by two cytochrome P450 monooxygenases, CYP734A1/BAS1 and CYP72C1/SOB7/CHI2/SHK1; CYP734A1/BAS1 inactivates CS and BL by means of C-26 hydroxylation. Here, we characterized CYP734A orthologs from Oryza sativa (rice). Overexpression of rice CYP734As in transgenic rice gave typical brassinosteroid-deficient phenotypes. These transformants were deficient in both the bioactive CS and its precursors downstream of the C-22 hydroxylation step. Consistent with this result, recombinant rice CYP734As utilized a range of C-22 hydroxylated brassinosteroid intermediates as substrates. In addition, rice CYP734As can catalyze hydroxylation and the second and third oxidations to produce aldehyde and carboxylate groups at C-26 in vitro. These results indicate that rice CYP734As are multifunctional, multisubstrate enzymes that control the endogenous bioactive brassinosteroid content both by direct inactivation of CS and by the suppression of CS biosynthesis by decreasing the levels of brassinosteroid precursors.     

Distinct binding of cholesterol and 5beta-cholestane-3alpha,7alpha,12alpha-triol to cytochrome P450 27A1: evidence from modeling and site-directed mutagenesis studies

Cytochrome P450 27A1 (P450 27A1 or CYP27A1) is an important enzyme that participates in different pathways of cholesterol degradation as well as in the activation of vitamin D(3). Several approaches were utilized to investigate how two physiological substrates, cholesterol and 5beta-cholestane-3alpha,7alpha,12alpha-triol, interact with CYP27A1. The enzyme active site was first probed spectrally by assessing binding of the two substrates and five substrate analogues followed by computer modeling and site-directed mutagenesis. The computer models suggest that the spatial positions and orientations of cholesterol and 5beta-cholestane-3alpha,7alpha,12alpha-triol are different in the enzyme active site. As a result, some of the active site residues interact with both substrates, although they are situated differently relative to each steroid, and some residues bind only one substrate. Mutation of the overlapping substrate-contact residues (W100, H103, T110, M301C, V367, I481, and V482) affected CYP27A1 binding and enzyme activity in a substrate-dependent manner and allowed identification of several important side chains. T110 is proposed to interact with the 12alpha-hydroxyl of 5beta-cholestane-3alpha,7alpha,12alpha-triol, whereas V367 seems to be crucial for correct positioning of the cholesterol C26 methyl group and for regioselective hydroxylation of this substrate. Distinct binding of the CYP27A1 substrates may provide insight into why phenotypic manifestations of cerebrotendinous xanthomatosis, a disease associated with CYP27A1 deficiency, are so diverse.     

Homology modeling and substrate binding study of human CYP4A11 enzyme

Although both bacterial CYP102 (P450BM3) and mammalian CYP4A isozymes share a common function as fatty acid hydroxylases, distinctly different preferred sites of oxidation are observed with the CYP102 performing the usual non-terminal hydroxylation or epoxidation and the CYP4A enzymes performing the unusual and enigmatic terminal hydroxylation. The origin of this unique product specificity in human CYP4A11 has been explored in this work, focusing on possible differences in the binding site architecture of the two isozymes as the cause. To this end, 3D model structures of the human CYP4A11 enzyme were built and compared to the X-ray structure of CYP102. The substrate-binding channel identified in CYP4A11 was found to have a much more sterically restricted active site than that in CYP102 that could cause limited access of long-chain fatty acid to the ferryl oxygen leading to the preferred omega-hydroxylation. Results of docking of a common substrate, lauric acid, into the binding site of both CYP4A11 and CYP102 and molecular dynamics simulations provided additional support for this hypothesis. Specifically, in the CYP4A11-lauric acid simulations, the omega hydrogens were closest to the ferryl oxygen most of the time. By contrast, in the CYP102-lauric acid complex, the substrate could penetrate further into the active site providing access of the non-terminal (omega-1, omega-2) positions to the ferryl oxygen. These results, taken together, have elucidated the origin of the unusual product specificity of CYP4A11 and illustrated the central role of binding site architecture in subtle modulation of function.     

Biotechnological Production of Caffeic Acid by Bacterial Cytochrome P450 CYP199A2

Caffeic acid is a biologically active molecule that has various beneficial properties, including antioxidant, anticancer, and anti-inflammatory activities. In this study, we explored the catalytic potential of a bacterial cytochrome P450, CYP199A2, for the biotechnological production of caffeic acid. When the CYP199A2 enzyme was reacted with p-coumaric acid, it stoichiometrically produced caffeic acid. The crystal structure of CYP199A2 shows that Phe at position 185 is situated directly above, and only 6.35 Å from, the heme iron. This F185 residue was replaced with hydrophobic or hydroxylated amino acids using site-directed mutagenesis to create mutants with novel and improved catalytic properties. In whole-cell assays with the known substrate of CYP199A2, 2-naphthoic acid, only the wild-type enzyme hydroxylated 2-naphthoic acid at the C-7 and C-8 positions, whereas all of the active F185 mutants exhibited a preference for C-5 hydroxylation. Interestingly, several F185 mutants (F185V, F185L, F185I, F185G, and F185A mutants) also acquired the ability to hydroxylate cinnamic acid, which was not hydroxylated by the wild-type enzyme. These results demonstrate that F185 is an important residue that controls the regioselectivity and the substrate specificity of CYP199A2. Furthermore, Escherichia coli cells expressing the F185L mutant exhibited 5.5 times higher hydroxylation activity for p-coumaric acid than those expressing the wild-type enzyme. By using the F185L whole-cell catalyst, the production of caffeic acid reached 15 mM (2.8 g/liter), which is the highest level so far attained in biotechnological production of this compound.     

Hydroxylation of DHEA and its analogues by Absidia coerulea AM93. Can an inducible microbial hydroxylase catalyze 7α- and 7β-hydroxylation of 5-ene and 5α-dihydro C19-steroids?

In this paper we focus on the course of 7-hydroxylation of DHEA, androstenediol, epiandrosterone, and 5α-androstan-3,17-dione by Absidia coerulea AM93. Apart from that, we present a tentative analysis of the hydroxylation of steroids in A. coerulea AM93. DHEA and androstenediol were transformed to the mixture of allyl 7-hydroxy derivatives, while EpiA and 5α-androstan-3,17-dione were converted mainly to 7α- and 7β-alcohols accompanied by 9α- and 11α-hydroxy derivatives. On the basis of (i) time course analysis of hydroxylation of the abovementioned substrates, (ii) biotransformation with resting cells at different pH, (iii) enzyme inhibition analysis together with (iv) geometrical relationship between the C–H bond of the substrate undergoing hydroxylation and the cofactor-bound activated oxygen atom, it is postulated that the same enzyme can catalyze the oxidation of C₇-H_α as well as C₇-H_β bonds in 5-ene and 5α-dihydro C₁₉-steroids. Correlations observed between the structure of the substrate and the regioselectivity of hydroxylation suggest that 7β-hydroxylation may occur in the normal binding enzyme-substrate complex, while 7α-hydroxylation—in the reverse inverted binding complex.     

Engineering of a water-soluble plant cytochrome P450, CYP73A1, and NMR-based orientation of natural and alternate substrates in the active site

CYP73A1 catalyzes cinnamic acid hydroxylation, a reaction essential for the synthesis of lignin monomers and most phenolic compounds in higher plants. The native CYP73A1, initially isolated from Jerusalem artichoke (Helianthus tuberosus), was engineered to simplify purification from recombinant yeast and improve solublity and stability in the absence of detergent by replacing the hydrophobic N terminus with the peptitergent amphipathic sequence PD1. Optimized expression and purification procedures yielded 4 mg engineered CYP73A1 L(-1) yeast culture. This water-soluble enzyme was suitable for 1H-nuclear magnetic resonance (NMR) investigation of substrate positioning in the active site. The metabolism and interaction with the enzyme of cinnamate and four analogs were compared by UV-visible and 1H-NMR analysis. It was shown that trans-3-thienylacrylic acid, trans-2-thienylacrylic acid, and 4-vinylbenzoic acid are good ligands and substrates, whereas trans-4-fluorocinnamate is a competitive inhibitor. Paramagnetic relaxation effects of CYP73A1-Fe(III) on the 1H-NMR spectra of cinnamate and analogs indicate that their average initial orientation in the active site is parallel to the heme. Initial orientation and distances of ring protons to the iron do not explain the selective hydroxylation of cinnamate in the 4-position or the formation of single products from the thienyl compounds. Position adjustments are thus likely to occur during the later steps of the catalytic cycle.     

Autodisplay of functional CYP106A2 in Escherichia coli

Cytochrome P450 enzymes catalyse a wide variety of reactions, including the hydroxylation and epoxidation of CC bonds, and dealkylation reactions. There is high interest in these reactions for biotechnology and pharmaceutical processes. Many P450s require membrane surroundings and have substrates that do not cross biological membranes. To circumvent these obstacles, CYP106A2 from Bacillus megaterium was expressed on the outer membrane of Escherichia coli cells by Autodisplay. Exposure on the surface was confirmed by a protease accessibility test and flow cytometry after immunolabelling. HPLC assays showed that 0.5 ml of cells displaying the enzyme (OD??? = 6) converted 9.13 μmol of deoxycorticosterone to 15β-OH-deoxycorticosterone within 1h. Imipramine and abietic acid were also accepted as substrates. The number of active enzyme molecules per cell was calculated to be 20,000. Surprisingly, surface-exposed CYP106A2 was active in E. coli BL21 without the external addition of the heme group. However, when CYP106A2 was expressed on the surface of an E. coli strain lacking the TolC channel protein (JW5503), enzymatic activity was almost completely abolished. The activity of CYP106A2 on the surface of E. coli JW5503 could be restored by the external addition of the heme group. This suggests, as has been reported before, that E. coli uses a TolC-dependent mechanism to export heme into the growth media, where it can be scavenged by a surface-displayed apoenzyme. Our results indicate that Autodisplay enables the functional surface display of P450 enzymes and provides a new platform to access their synthetic potential.     

Fungal laccases as green catalysts for dye synthesis

Laccases from different sources catalyse oxidation of various phenolic and aromatic compounds to products that very often are colourful and may be used as dyes, especially in the textile industry. They catalyse not only catabolic processes such as depolymerisation and degradation but can also carry out various dimerization, oligomerization, and polymerization reactions of some hundred aromatic substrates that synthesize new molecules with valuable functions. Because of their versatile biochemical properties, high protein stability, breadth of substrate spectrum, laccases are the key enzymes having applications in biotechnological processes as eco-friendly biocatalyst. This review refers to the natural abilities of laccases to synthesize colour products with respect to the type of the enzymatic reaction, catalyst characterization and possible use of these colour products as dyestuffs.     

Sequence-based screening for self-sufficient P450 monooxygenase from a metagenome library

AIMS: Cytochrome P450 monooxygenases (CYPs) are useful catalysts for oxidation reactions. Self-sufficient CYPs harbour a reductive domain covalently connected to a P450 domain and are known for their robust catalytic activity with great potential as biocatalysts. In an effort to expand genetic sources of self-sufficient CYPs, we devised a sequence-based screening system to identify them in a soil metagenome. METHODS AND RESULTS: We constructed a soil metagenome library and performed sequence-based screening for self-sufficient CYP genes. A new CYP gene, syk181, was identified from the metagenome library. Phylogenetic analysis revealed that SYK181 formed a distinct phylogenic line with 46% amino-acid-sequence identity to CYP102A1 which has been extensively studied as a fatty acid hydroxylase. The heterologously expressed SYK181 showed significant hydroxylase activity towards naphthalene and phenanthrene as well as towards fatty acids. CONCLUSIONS: Sequence-based screening of metagenome libraries is expected to be a useful approach for searching self-sufficient CYP genes. The translated product of syk181 shows self-sufficient hydroxylase activity towards fatty acids and aromatic compounds. SIGNIFICANCE AND IMPACT OF THE STUDY: SYK181 is the first self-sufficient CYP obtained directly from a metagenome library. The genetic and biochemical information on SYK181 are expected to be helpful for engineering self-sufficient CYPs with broader catalytic activities towards various substrates, which would be useful for bioconversion of natural products and biodegradation of organic chemicals.     

Biotransformation XXXIX. Metabolism of testosterone, androstenedione, progesterone and testosterone derivatives in Absidia coerulea culture

The strain of Absidia coerulea was used to investigate the transformations of testosterone, androstenedione, progesterone and testosterone derivatives with additional C1–C2 double bond and/or 17-methyl group. All the examined substrates were transformed, mainly hydroxylated. It was found that the position and stereochemistry of the introduced hydroxyl group, as well as the yield of products, depended on the structure of the substrate. The first three substrates (hormones) underwent hydroxylation at C-14, and additional hydroxylation at 7 was observed in progesterone. The presence of the double bond (C1–C2) in 1-dehydrotestosterone did not influence the position of hydroxylation, but the product with additional C14–C15 double bond (at the same site as hydroxylation) was formed. 17-Methyltestosterone was hydroxylated at the 7 position, and also the dehydrogenated product (at the same site, with C6–C7 double bond) was obtained. The testosterone derivative with both C1–C2 double bond and 17-methyl group underwent hydroxylation at the 7 or 11β position, and a little amount of 14, 15 epoxide was formed.     

Homology modeling and substrate binding study of human CYP2C18 and CYP2C19 enzymes

It is well established that the variable binding-site architecture and composition of the P450 metabolizing heme proteins are major modulators of substrate and product specificity. Even the three closely related human liver isozymes, CYP2C9, CYP2C18, and CYP2C19, do not share all substrates and do not always lead to the same preferred hydroxylation products. The lack of knowledge of their three-dimensional (3D) structures has hindered efforts to understand the differences in their specificities. Building on previous work for the CYP2C9 enzyme, 3D models of CYP2C18 and 2C19 have been constructed and validated by computational methods developed and tested in our laboratory. They were used to characterize explicit enzyme-substrate complexes using the isoform-specific substrates progesterone and (S)-mephenytoin for 2C19 and 2-[2,3-dichloro-4-(3-hydroxypropyloxy)benzoyl]thiophene for 2C18. The results allowed both common and unique binding-site residues to be identified in each model. The calculated preferred hydroxylation site was obtained for each substrate and was found to be consistent with experimental observation. Comparisons were made among the 2C9, 2C18, and 2C19 model binding sites to investigate the subtle differences among them. These models can be used as structure-based guides for mutagenesis studies and screening of potential pharmaceuticals or toxins.