Abstract
Cytochrome P450BM3-F87G catalyzed the oxidative defluorination of 4-fluorophenol, followed by reduction of the resulting benzoquinone to hydroquinone via the NADPH P450-reductase activity of the enzyme. The k cat and K m for this reaction were 71 ± 5 min−1 and 9.5 ± 1.3 mM, respectively. Co-incubation of the reaction mixture with long chain aldehydes stimulated the defluorination reaction, with the 2,3-unsaturated aldehyde, 2-decenal producing a 12-fold increase in catalytic efficiency. At 150 μM aldehyde, k cat increased to 158 ± 4, while K m decreased to 1.8 ± 0.2. The effects of catalase, glutathione and ascorbate on the reaction were all consistent with a direct oxygen insertion mechanism, as opposed to a radical mechanism. The study demonstrates the potential use of P450BM3 mutants in oxidative defluorination reactions, and characterizes the novel stimulatory action of straight chain aldehydes on this activity.
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References
Harvey PJ, Campanella BF, Castro PML, Harms H, Lichtfouse E, Schäffner AR, Smrcek S, Werck-Reichhart D (2002) Phytoremediation of polyaromatic hydrocarbons, anilines and phenols. Environ Sci Pollut Res 9:29–47
Key BD, Howell RD, Criddle CS (1997) Fluorinated organics in the biosphere. Environ Sci Technol 31:2445–2454
Lau SS, Hill BA, Highet RJ, Monks TJ (1988) Sequential oxidation and glutathione addition to 1,4-benzoquinone: correlation of toxicity with increased glutathione substitution. Mol Pharmacol 34:829–836
Meharg AA, Wright J, Osborn D (2000) Chlorobenzenes in rivers draining industrial catchments. Sci Total Environ 251:243–253
Murphy CD (2007) Fluorophenol oxidation by a fungal chloroperoxidase. Biotechnol Lett 29:45–49
Ohe T, Mashino T, Hirobe M (1997) Substituent elimination form p-substituted phenols by cytochrome P450 ipso substitution by the activated oxygen atom of the activated species. Drug Metab Dispos 25:116–122
Osborne RL, Raner GM, Hager LP, Dawson JH (2006) C. fumago chloroperoxidase is also a dehaloperoxidase: oxidative dehalogenation of halophenols. J Amer Chem Soc 128:1036–1037
Osborne RL, Coggins MK, Raner GM, Walla M, Dawson JH (2009) The mechanism of oxidative halophenol dehalogenation by Amphitrite ornata dehaloperoxidase is initiated by H2O2 binding and involves two consecutive one-electron steps: role of ferryl intermediates. Biochemistry 48:4231–4238
Osman AM, Boeren S, Veeger C, Rietjens IMCM (1997) MP8-Dependent oxidative dehalogenation: evidence for the direct formation of 1,4-benzoquinone from 4-fluorophenol by a peroxidase-type of reaction pathway. Chem Biol Interact 104:147–164
Raner GM, Hatchell AJ, Morton PE, Ballou DP, Coon MJ (2000) Stopped-flow spectrophotometric analysis of intermediates in the peroxo-dependent inactivation of cytochrome p450 by aldehydes. J Inorg Biochem 81:153–160
Raner GM, Hatchell JA, Dixon MU, Joy T, Haddy AE, Johnston ER (2002) Regioselective peroxo-dependent heme alkylation in p450bm3-f87g by aromatic aldehydes: effects of alkylation on catalysis. Biochemistry 41:9601–9610
Rietjens IMCM, Vervoort J (1992) A new hypothesis for the mechanism for the cytochrome P-450 dependent aerobic conversion of hexahalogenated benzenes to pentahalogenated phenols. Chem Res Toxicol 5:10–19
Schwaneberg U, Schmidt-Dannert C, Schmit J, Schmid RD (1999) A continuous spectrophotometric assay for P450BM-3, a fatty acid hydroxylating enzyme, and its mutant F87A. Anal Biochem 269:359–366
Szatkowski L, Thompson MK, Kaminski R, Franzen S, Dybala-Defratyka A (2011) Oxidative dechlorination of halogenated phenols catalyzed by two distinct enzymes: horseradish peroxidase and dehaloperoxidase. Arch Biochem Biophys 508:22–32
Vatsis KP, Coon MJ (2002) Ipso substitution by Cytochrome P450 with conversion of the p-hydroxybenzene derivatives to hydroquinone: evidence for hydroperoxo-iron as the active oxygen species. Arch Biochem Biophys 397:119–129
Acknowledgments
Funding for this research was provided by The National Science Foundation (#0414301), Research Corporation (CC4924) and The American Chemical Society Petroleum Research Fund (41094-UFS and 37796-B4) to G.M.R.
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Harkey, A., Kim, HJ., Kandagatla, S. et al. Defluorination of 4-fluorophenol by cytochrome P450BM3-F87G: activation by long chain fatty aldehydes. Biotechnol Lett 34, 1725–1731 (2012). https://doi.org/10.1007/s10529-012-0957-9
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DOI: https://doi.org/10.1007/s10529-012-0957-9