Abstract
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.
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References
Abdulmughni A, Jóźwik IK, Putkaradze N, Brill E, Zapp J, Thunnissen AMWH, Hannemann F, Bernhardt R (2017) Characterization of cytochrome P450 CYP109E1 from Bacillus megaterium as a novel vitamin D3 hydroxylase. J Biotechnol 243:38–47. https://doi.org/10.1016/j.jbiotec.2016.12.023
Ba L, Li P, Zhang H, Duan Y, Lin Z (2013) Semi-rational engineering of cytochrome P450sca-2 in a hybrid system for enhanced catalytic activity: insights into the important role of electron transfer. Biotechnol Bioeng 110:2815–2825. https://doi.org/10.1002/bit.24960
Barg H, Malten M, Jahn M, Jahn D (2005) Protein and vitamin production in Bacillus megaterium. In: Barredo JL (ed) Microbial processes and products. Humana Press, New York, pp 205–223
Bell SG, Wong LL (2007) P450 enzymes from the bacterium Novosphingobium aromaticivorans. Biochem Biophys Res Commun 360(3):666–672. https://doi.org/10.1016/j.bbrc.2007.06.119
Berg A, Rafter JJ (1981) Studies on the substrate specificity and inducibility of cytochrome P-450meg. Biochem J 196:781–786
Bernhardt R (2006) Cytochromes P450 as versatile biocatalysts. J Biotechnol 124:128–145. https://doi.org/10.1016/j.jbiotec.2006.01.026
Bernhardt R, Urlacher VB (2014) Cytochromes P450 as promising catalysts for biotechnological application: chances and limitations. Appl Microbiol Biotechnol 98:6185–6203. https://doi.org/10.1007/s00253-014-5767-7
Biedendieck R, Malten M, Barg H, Bunk B, Martens JH, Deery E, Leech H, Warren MJ, Jahn D (2010) Metabolic engineering of cobalamin (vitamin B12) production in Bacillus megaterium. Microb Biotechnol 3:24–37. https://doi.org/10.1111/j.1751-7915.2009.00125.x
Bleif S, Hannemann F, Zapp J, Hartmann D, Jauch J, Bernhardt R (2011) A new Bacillus megaterium whole-cell catalyst for the hydroxylation of the pentacyclic triterpene 11-keto-β-boswellic acid (KBA) based on a recombinant cytochrome P450 system. Appl Microbiol Biotechnol 93:1135–1146. https://doi.org/10.1007/s00253-011-3467-0
Brill E, Hannemann F, Zapp J, Brüning G, Jauch J, Bernhardt R (2014) A new cytochrome P450 system from Bacillus megaterium DSM319 for the hydroxylation of 11-keto-β-boswellic acid (KBA). Appl Microbiol Biotechnol 98:1701–1717. https://doi.org/10.1007/s00253-013-5029-0
Brown AJ (2007) Cholesterol, statins and cancer. Clin Exp Pharmacol Physiol 34:135–141. https://doi.org/10.1111/j.1440-1681.2007.04565.x
Çelik A, Flitsch SL, Turner NJ (2005) Efficient terpene hydroxylation catalysts based upon P450 enzymes derived from Actinomycetes. Org Biomol Chem 3:2930–2934. https://doi.org/10.1039/b506159h
Choudhary MI, Musharraf SG, Khan MTH, Abdelrahman D, Parvez M, Shaheen F, Rahman A-u (2003) Microbial transformation of isolongifolen-4-one. Helv Chim Acta 86:3450–3460. https://doi.org/10.1002/hlca.200390289
Endo A, Hasumi K (1993) HMG-CoA reductase inhibitors. Nat Prod Rep 10:541–550. https://doi.org/10.1039/NP9931000541
Eppinger M, Bunk B, Johns MA, Edirisinghe JN, Kutumbaka KK, Koenig SSK, Creasy HH, Rosovitz MJ, Riley DR, Daugherty S, Martin M, Elbourne LD, Paulsen I, Biedendieck R, Braun C, Grayburn S, Dhingra S, Lukyanchuk V, Ball B, Ul-Qamar R, Seibel J, Bremer E, Jahn D, Ravel J, Vary PS (2011) Genome sequences of the biotechnologically important Bacillus megaterium strains QM B1551 and DSM319. J Bacteriol 193:4199–4213. https://doi.org/10.1128/JB.00449-11
Fisher MT, Sligar SG (1985) Control of heme protein redox potential and reduction rate: linear free energy relation between potential and ferric spin state equilibrium. J Am Chem Soc 107(17):5018–5019
Fulco AJ (1991) P450BM-3 and other inducible bacterial P450 cytochromes: biochemistry and regulation. Annu Rev Pharmacol Toxicol 31:177–203. https://doi.org/10.1146/annurev.pa.31.040191.001141
Furuya T, Shibata D, Kino K (2009) Phylogenetic analysis of Bacillus P450 monooxygenases and evaluation of their activity towards steroids. Steroids 74:906–912. https://doi.org/10.1016/j.steroids.2009.06.005
Gazzerro P, Proto MC, Gangemi G, Malfitano AM, Ciaglia E, Pisanti S, Santoro A, Laezza C, Bifulco M (2012) Pharmacological actions of statins: a critical appraisal in the management of cancer. Pharmacol Rev 64:102–146. https://doi.org/10.1124/pr.111.004994
Girhard M, Klaus T, Khatri Y, Bernhardt R, Urlacher VB (2010) Characterization of the versatile monooxygenase CYP109B1 from Bacillus subtilis. Appl Microbiol Biotechnol 87:595–607. https://doi.org/10.1007/s00253-010-2472-z
Gliszczyńska A, Łysek A, Janeczko T, Świtalska M, Wietrzyk J, Wawrzeńczyk C (2011) Microbial transformation of (+)-nootkatone and the antiproliferative activity of its metabolites. Bioorg Med Chem 19:2464–2469. https://doi.org/10.1016/j.bmc.2011.01.062
Gliszczyńska A, Gładkowski W, Dancewicz K, Gabryś B, Szczepanik M (2016) Transformation of β-damascone to (+)-(S)-4-hydroxy-β-damascone by fungal strains and its evaluation as a potential insecticide against aphids Myzus persicae and lesser mealworm Alphitobius diaperinus Panzer. Catal Commun 80:39–43. https://doi.org/10.1016/j.catcom.2016.03.018
Hall EA, Bell SG (2014) The efficient and selective biocatalytic oxidation of norisoprenoid and aromatic substrates by CYP101B1 from Novosphingobium aromaticivorans DSM12444. RSC Adv 5:5762–5773. https://doi.org/10.1039/c4ra14010a
Honeychurch MJ, Hill AO, Wong LL (1999) The thermodynamics and kinetics of electron transfer in the cytochrome P450cam enzyme system. FEBS Lett 451(3):351–353
Janocha S, Schmitz D, Bernhardt R (2015) Terpene hydroxylation with microbial cytochrome P450 monooxygenases. Adv Biochem Eng Biotechnol 148:215–250. https://doi.org/10.1007/10_2014_296
Janocha S, Carius Y, Hutter M, Lancaster CRD, Bernhardt R (2016) Crystal structure of CYP106A2 in substrate-free and substrate-bound form. Chembiochem 17(9):852–860. https://doi.org/10.1002/cbic. 201500524
Jóźwik IK, Kiss FM, Gricman Ł, Abdulmughni A, Brill E, Zapp J, Pleiss J, Bernhardt R, Thunnissen AWH (2016) Structural basis of steroid binding and oxidation by the cytochrome P450 CYP109E1 from Bacillus megaterium. FEBS J 283:4128–4148. https://doi.org/10.1111/febs.13911
Kandel SE, Wienkers LC, Lampe JN (2014) Cytochrome P450 enzyme metabolites in lead discovery and development. Annu Rep Med Chem 49:347–359. https://doi.org/10.1016/B978-0-12-800167-7.00022-5
Katagiri M, Ganguli BN, Gunsalus IC (1968) A soluble cytochrome P-450 functional in methylene hydroxylation. J Biol Chem 243(12):3543–3546
Kellick KA, Bottorff M, Toth PP (2014) A clinician’s guide to statin drug-drug interactions. J Clin Lipidol 8:30–46. https://doi.org/10.1016/j.jacl.2014.02.010
Khatri Y, Girhard M, Romankiewicz A, Ringle M, Hannemann F, Urlacher VB, Hutter MC, Bernhardt R (2010) Regioselective hydroxylation of norisoprenoids by CYP109D1 from Sorangium cellulosum So ce56. Appl Microbiol Biotechnol 88:485–495. https://doi.org/10.1007/s00253-010-2756-3
Khatri Y, Ringle M, Lisurek M, von Kries JP, Zapp J, Bernhardt R (2016) Substrate hunting for the myxobacterial CYP260A1 revealed new 1α-hydroxylated products from C-19 steroids. Chembiochem 17(1):90–101. https://doi.org/10.1002/cbic.201500420
Khera S, Hu N (2013) Generation of statin drug metabolites through electrochemical and enzymatic oxidations. Anal Bioanal Chem 405:6009–6018. https://doi.org/10.1007/s00216-013-7021-z
Kim KH, Kang JY, Kim DH, Park SH, Park SH, Kim D, Park KD, Lee YJ, Jung HC, Pan JG, Ahn T, Yun CH (2011) Generation of human chiral metabolites of simvastatin and lovastatin by bacterial CYP102A1 mutants. Drug Metab Dispos 39(1):140–150. https://doi.org/10.1124/dmd.110.036392
Kiss FM, Schmitz D, Zapp J, Dier TKF, Volmer DA, Bernhardt R (2015) Comparison of CYP106A1 and CYP106A2 from Bacillus megaterium—identification of a novel 11-oxidase activity. Appl Microbiol Biotechnol 99:8495–8514. https://doi.org/10.1007/s00253-015-6563-8
Korneli C, Biedendieck R, David F, Jahn D, Wittmann C (2013) High yield production of extracellular recombinant levansucrase by Bacillus megaterium. Appl Microbiol Biotechnol 97:3343–3353. https://doi.org/10.1007/s00253-012-4567-1
Kulpreecha S, Boonruangthavorn A, Meksiriporn B, Thongchul N (2009) Inexpensive fed-batch cultivation for high poly(3-hydroxybutyrate) production by a new isolate of Bacillus megaterium. J Biosci Bioeng 107:240–245. https://doi.org/10.1016/j.jbiosc.2008.10.006
Lamon-Fava S (2013) Statins and lipid metabolism: an update. Curr Opin Lipidol 24:221–226. https://doi.org/10.1097/MOL.0b013e3283613b8b
Lisurek M, Kang MJ, Hartmann RW, Bernhardt R (2004) Identification of monohydroxy progesterones produced by CYP106A2 using comparative HPLC and electrospray ionisation collision-induced dissociation mass spectrometry. Biochem Biophys Res Commun 319:677–682
Litzenburger M, Bernhardt R (2016) Selective oxidation of carotenoid-derived aroma compounds by CYP260B1 and CYP267B1 from Sorangium cellulosum So ce56. Appl Microbiol Biotechnol 100:4447–4457. https://doi.org/10.1007/s00253-015-7269-7
Luthra A, Denisov IG, Sligar SG (2011) Spectroscopic features of cytochrome P450 reaction intermediates. Arch Biochem Biophys 507(1):26–35. https://doi.org/10.1016/j.abb.2010.12.008
Ma M, Bell SG, Yang W, Hao Y, Rees NH, Bartlam M, Zhou W, Wong LL, Rao Z (2011) Structural analysis of CYP101C1 from Novosphingobium aromaticivorans DSM12444. Chembiochem 12:88–99
Malten M, Hollmann R, Deckwer WD, Jahn D (2005) Production and secretion of recombinant Leuconostoc mesenteroides dextransucrase DsrS in Bacillus megaterium. Biotechnol Bioeng 89:206–218. https://doi.org/10.1002/bit.20341
Matsuoka T, Miyakoshi S, Tanzawa K, Nakahara K, Hosobuchi M, Serizawa N (1989) Purification and characterization of cytochrome P-450sca from Streptomyces carbophilus. Eur J Biochem 184:707–713. https://doi.org/10.1111/j.1432-1033.1989.tb15070.x
McLean KJ, Hans M, Meijrink B, van Scheppingen WB, Vollebregt A, Tee KL, van der Laan JM, Leys D, Munro AW, van den Berg MA (2015) Single-step fermentative production of the cholesterol-lowering drug pravastatin via reprogramming of Penicillium chrysogenum. Proc Natl Acad Sci U S A 112:2847–2852. https://doi.org/10.1073/pnas.1419028112
Michaels BC, Ruettinger RT, Fulco AJ (1980) Hydration of 9,10-epoxypalmitic acid by a soluble Enzyme from Bacillus megaterium. Biochem Biophys Res Commun 92(4):1189–1195
Milhim M, Putkaradze N, Abdulmughni A, Kern F, Hartz P, Bernhardt R (2016) Identification of a new plasmid-encoded cytochrome P450 CYP107DY1 from Bacillus megaterium with a catalytic activity towards mevastatin. J Biotechnol 240:68–75. https://doi.org/10.1016/j.jbiotec.2016.11.002
Miura Y, Fulco AJ (1975) ω-1, ω-2 and ω-3 hydroxylation of long-chain fatty acids, amides and alcohols by a soluble enzyme system from Bacillus megaterium. Biochim Biophys Acta 388:305–317
More GP, Bhat SV (2013) Facile lipase catalysed syntheses of (S)-(+)-4-hydroxy-β-ionone and (S)-(+)-4-hydroxy-β-damascone: chiral flavorants and synthons. Tetrahedron Lett 54:4148–4149. https://doi.org/10.1016/j.tetlet.2013.05.089
Morris GM, Huey R, Lindstrom W, Sanner MF, Belew RK, Goodsell DS, Olson AJ (2009) AutoDock4 and AutoDockTools4: automated docking with selective receptor flexibility. J Comput Chem 30(16):2785–2791. https://doi.org/10.1002/jcc.21256
Narhi LO, Fulco AJ (1987) Identification and characterization of two functional domains in cytochrome P-450BM-3, a catalytically self-sufficient monooxygenase induced by barbiturates in Bacillus megaterium. J Biol Chem 262:6683–6690
Omura T, Sato R (1964) The carbon monoxide-binding pigment of liver microsomes. I. Evidence for its hemoprotein nature. J Biol Chem 239:2370–2378
Peterson JA, Lu JY, Geisselsoder J, Graham-Lorence S, Carmona C, Witney F, Lorence MC (1992) Cytochrome P-450terp. Isolation and purification ofthe protein and cloning and sequencing of its operon. J Biol Chem 267(20):14193–14203
Poulos TL, Raag R (1992) Cytochrome P450cam: crystallography, oxygen activation, and electron transfer. FASEB J 6(2):674–679
Putkaradze N, Kiss FM, Schmitz D, Zapp J, Hutter MC, Bernhardt R (2017) Biotransformation of prednisone and dexamethasone by cytochrome P450 based systems—identification of new potential drug candidates. J Biotechnol 242:101–110. https://doi.org/10.1016/j.jbiotec.2016.12.011
Rauschenbach R, Isernhagen M, Noeske-Jungblut C, Boidol W, Siewert G (1993) Cloning sequencing and expression of the gene for cytochrome P450meg, the steroid-15β-monooxygenase from Bacillus megaterium ATCC 13368. Mol Gen Genet 241:170–176
Rygus T, Hillen W (1992) Catabolite repression of the xyl operon in Bacillus megaterium. J Bacteriol 174:3049–3055
Sagara Y, Wada A, Takata Y, Waterman MR, Sekimizu K, Horiuchi T (1993) Direct expression of adrenodoxin reductase in Escherichia coli and the functional characterization. Biol Pharm Bull 16:627–630
Sakaki T (2012) Practical application of cytochrome P450. Biol Pharm Bull 35:844–849
Sakata K, Oda Y, Miyazawa M (2010) Suppression of SOS-inducing activity of chemical mutagens by metabolites from microbial transformation of (−)-isolongifolene. J Agric Food Chem 58:2164–2167. https://doi.org/10.1021/jf903651c
Sanner MF (1999) Python: a programming language for software integration and development. J Mol Graph Modell 17(1):57–61
Schenkman JB, Jansson I (1998) Spectral analyses of cytochromes P450. In: Phillips I, Shephard EA (eds) Cytochrome P450 protocols. Humana Press, New York, pp 25–34
Schifrin A, Litzenburger M, Ringle M, Ly TTB, Bernhardt R (2015) New sesquiterpene oxidations with CYP260A1 and CYP264B1 from Sorangium cellulosum So ce56. Chembiochem 16:2624–2632. https://doi.org/10.1002/cbic.201500417
Schmitz D, Zapp J, Bernhardt R (2012) Hydroxylation of the triterpenoid dipterocarpol with CYP106A2 from Bacillus megaterium. FEBS J 279:1663–1674. https://doi.org/10.1111/j.1742-4658.2012. 08503.x
Schmitz D, Zapp J, Bernhardt R (2014) Steroid conversion with CYP106A2—production of pharmaceutically interesting DHEA metabolites. Microb Cell Factories 13:81. https://doi.org/10.1186/1475-2859-13-81
Simgen B, Contzen J, Schwarzer R, Bernhardt R, Jung C (2000) Substrate binding to 15β-hydroxylase (CYP106A2) probed by FT infrared spectroscopic studies of the iron ligand CO stretch vibration. Biochem Biophys Res Commun 269:737–742. https://doi.org/10.1006/bbrc.2000.2348
Sligar SG, Gunsalus IC (1976) A thermodynamic model of regulation: modulation of redox equilibria in camphor monoxygenase. Proc Natl Acad Sci 73(4):1078–1082
Sono M, Roach MP, Coulter ED, Dawson JH (1996) Heme-containing oxygenases. Chem Rev 96:2841–2888
Sowden RJ, Yasmin S, Rees NH, Bell SG, Wong LL (2005) Biotransformation of the sesquiterpene (+)-valencene by cytochrome P450cam and P450BM-3. Org Biomol Chem 3:57–64. https://doi.org/10.1039/b413068e
Stammen S, Müller BK, Korneli C, Biedendieck R, Gamer M, Franco-Lara E, Jahn D (2010) High-yield intra- and extracellular protein production using Bacillus megaterium. Appl Environ Microbiol 76:4037–4046. https://doi.org/10.1128/AEM.00431-10
Stokker GE (1994) Synthesis of the 3′(S)-hydroxy derivative of simvastatin. Bioorg Med Chem Lett 4:1767–1770. https://doi.org/10.1016/S0960-894X(00)80377-7
Tang YF, Xu JH, Ye Q, Schulze B (2001) Biocatalytic preparation of (S)-phenyl glycidyl ether using newly isolated Bacillus megaterium ECU1001. J Mol Catal B Enzym 13:61–68. https://doi.org/10.1016/S1381-1177(00)00230-7
Uhlmann H, Beckert V, Schwarz D, Bernhardt R (1992) Expression of bovine adrenodoxin in E. coli and site-directed mutagenesis of /2FE-2S/ cluster ligands. Biochem Biophys Res Commun 188:1131–1138
Ullah AJ, Murray RI, Bhattacharyya PK, Wagner GC, Gunsalus IC (1990) Protein components of a cytochrome P-450 linalool 8-methyl hydroxylase. J Biol Chem 265(3):1345–1351
Urlacher VB, Lutz-Wahl S, Schmid RD (2004) Microbial P450 enzymes in biotechnology. Appl Microbiol Biotechnol 64:317–325. https://doi.org/10.1007/s00253-003-1514-1
Urlacher VB, Makhsumkhanov A, Schmid RD (2006) Biotransformation of beta-ionone by engineered cytochrome P450 BM-3. Appl Microbiol Biotechnol 70:53–59. https://doi.org/10.1007/s00253-005-0028-4
Vary PS, Biedendieck R, Fuerch T, Meinhardt F, Rohde M, Deckwer WD, Jahn D (2007) Bacillus megaterium—from simple soil bacterium to industrial protein production host. Appl Microbiol Biotechnol 76:957–967. https://doi.org/10.1007/s00253-007-1089-3
Virus C, Lisurek M, Simgen B, Hannemann F, Bernhardt R (2006) Function and engineering of the 15β-hydroxylase CYP106A2. Biochem Soc Trans 34:1215–1218. https://doi.org/10.1042/BST0341215
Williams JW, Morrison JF (1979) The kinetics of reversible tight-binding inhibition. Methods Enzymol 63:437–467
Wittchen KD, Meinhardt F (1995) Inactivation of the major extracellular protease from Bacillus megaterium DSM319 by gene replacement. Appl Microbiol Biotechnol 42:871–877. https://doi.org/10.1007/ BF00191184
Zhang Z, Sheng Y, Jiang K, Wang Z, Zheng Y, Zhu Q (2010) Bio-resolution of glycidyl (o, m, p)-methylphenyl ethers by Bacillus megaterium. Biotechnol Lett 32:513–516. https://doi.org/10.1007/s10529-009-0181-4
Acknowledgements
The authors thank Birgit Heider-Lips for the purification of AdR and Adx4-108, Dr. Josef Zapp for the NMR measurements, and Ghamdan Beshr from the Helmholtz Institute for Pharmaceutical Research Saarland (HIPS) for LC-MS measurements.
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Putkaradze, N., Litzenburger, M., Abdulmughni, A. et al. CYP109E1 is a novel versatile statin and terpene oxidase from Bacillus megaterium . Appl Microbiol Biotechnol 101, 8379–8393 (2017). https://doi.org/10.1007/s00253-017-8552-6
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DOI: https://doi.org/10.1007/s00253-017-8552-6