Abstract
Baeyer–Villiger monooxygenases represent useful biocatalytic tools, as they can catalyze reactions which are difficult to achieve using chemical means. However, only a limited number of these atypical monooxygenases are available in recombinant form. Using a recently described protein sequence motif, a putative Baeyer–Villiger monooxygenase (BVMO) was identified in the genome of the thermophilic actinomycete Thermobifida fusca. Heterologous expression of the respective protein in Escherichia coli and subsequent enzyme characterization showed that it indeed represents a BVMO. The NADPH-dependent and FAD-containing monooxygenase is active with a wide range of aromatic ketones, while aliphatic substrates are also converted. The best substrate discovered so far is phenylacetone (kcat = 1.9 s−1, KM = 59 μM). The enzyme exhibits moderate enantioselectivity with α-methylphenylacetone (enantiomeric ratio of 7). In addition to Baeyer–Villiger reactions, the enzyme is able to perform sulfur oxidations. Different from all known BVMOs, this newly identified biocatalyst is relatively thermostable, displaying an activity half-life of 1 day at 52°C. This study demonstrates that, using effective annotation tools, genomes can efficiently be exploited as a source of novel BVMOs.
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References
Abokitse K, Hummel W (2003) Cloning, sequence analysis, and heterologous expression of the gene encoding a (S)-specific alcohol dehydrogenase from Rhodococcus erythropolis DSM 43297. Appl Microbiol Biotechnol 62:380–386
Alphand V, Carrea G, Wohlgemuth R, Furstoss R, Woodley JM (2003) Towards large-scale synthetic applications of Baeyer–Villiger monooxygenases. Trends Biotechnol 21:318–323
Antoine E, Rolland JL, Raffin JP, Dietrich J (1999) Cloning and over-expression in Escherichia coli of the gene encoding NADPH group III alcohol dehydrogenase from Thermococcus hydrothermalis. Characterization and comparison of the native and the recombinant enzymes. Eur J Biochem 264:880–889
Beilen JB van, Duetz WA, Schmid A, Witholt B (2003a) Practical issues in the application of oxygenases. Trends Biotechnol 21:170–177
Beilen JB van, Mourlane F, Seeger MA, Kovac J, Li Z, Smits TH, Fritsche U, Witholt B (2003b) Cloning of Baeyer–Villiger monooxygenases from Comamonas, Xanthobacter and Rhodococcus using polymerase chain reaction with highly degenerate primers. Environ Microbiol 5:174–182
Brzostowicz PC, Walters DM, Thomas SM, Nagarajan V, Rouviere PE (2003) mRNA differential display in a microbial enrichment culture: simultaneous identification of three cyclohexanone monooxygenases from three species. Appl Environ Microbiol 69:334–342
Chen YC, Peoples OP, Walsh CT (1988) Acinetobacter cyclohexanone monooxygenase: gene cloning and sequence determination. J Bacteriol 170:781–789
Flitsch S, Grogan G (2002) Baeyer-Villiger monooxygenases. In: Drauz K, Waldmann H (eds) Enzyme catalysis in organic synthesis. Wiley-VCH, Weinheim, pp 1202–1245
Fraaije MW, Kamerbeek NM, Berkel WJH van, Janssen DB (2002) Identification of a Baeyer–Villiger monooxygenase sequence motif. FEBS Lett 518:43–47
Fraaije MW, Kamerbeek NM, Heidekamp AJ, Fortin R, Janssen DB (2004) The prodrug activator EtaA from Mycobacterium tuberculosis is a Baeyer–Villiger monooxygenase. J Biol Chem 279:3354–3360
Hefti MH, Vervoort J, Berkel WJH van (2003) Deflavination and reconstitution of flavoproteins. Eur J Biochem 270:4227–4242
Hilker I, Gutierrez MC, Alphand V, Wohlgemuth R, Furstoss R (2004) Microbiological transformations 57. Facile and efficient resin-based in situ SFPR preparative-scale synthesis of an enantiopure “unexpected” lactone regioisomer via a Baeyer–Villiger oxidation process. Org Lett 6:1955–1958
Iwaki N, Hasegawa Y, Wang S, Kayser MM, Lau PCK (2002) Cloning and characterization of a gene cluster involved in cyclopentanol metabolism in Comamonas sp. strain NCIMB 9872 and biotransformations effected by Escherichia coli-expressed cyclopentanone 1,2-monooxygenase. Appl Environ Microbiol 68:5671–5684
Kamerbeek NM, Moonen MJ, Van Der Ven JG, Van Berkel WJH, Fraaije MW, Janssen DB (2001) 4-Hydroxyacetophenone monooxygenase from Pseudomonas fluorescens ACB. A novel flavoprotein catalyzing Baeyer–Villiger oxidation of aromatic compounds. Eur J Biochem 268:2547–2557
Kamerbeek NM, Janssen DB, Berkel WJH van, Fraaije MW (2003a) Baeyer–Villiger monooxygenases, an emerging family of flavin-dependent biocatalysts. Adv Synth Catal 345:1–12
Kamerbeek NM, Olsthoorn AJJ, Fraaije MW, Janssen DB (2003b) Substrate specificity and enantioselectivity of 4-hydroxyacetophenone monooxygenase. Appl Environ Microbiol 69:419–426
Kelly DR (2000) Enantioselective Baeyer–Villiger reactions. Chim Oggi 18:333–337
Kobayashi T, Higuchi S, Kimura K, Kudo T, Horikoshi K (1995) Properties of glutamate dehydrogenase and its involvement in alanine production in a hyperthermophilic archaeon, Thermococcus profundus. J Biochem 118:587–592
Kostichka K, Thomas SM, Gibson KJ, Nagarajan V, Cheng Q (2001) Cloning and characterization of a gene cluster for cyclododecanone oxidation in Rhodococcus ruber SC1. J Bacteriol 183:6478–6486
Mihovilovic MD, Muller B, Stanetty P (2002) Monooxygenase-mediated Baeyer–Villiger oxidations. Eur J Org Chem 22:3711–3730
Morii S, Sawamoto S, Yamauchi Y, Miyamoto M, Iwami M, Itagaki E (1999) Steroid monooxygenase of Rhodococcus rhodochrous: sequencing of the genomic DNA, and hyperexpression, purification, and characterization of the recombinant enzyme. J Biochem 126:624–631
Poulsen LL, Ziegler DM (1995) Multisubstrate flavin-containing monooxygenases: applications of mechanism to specificity. Chem Biol Interact 96:57–73
Roberts SM, Wan PWH (1998) Enzyme-catalysed Baeyer–Villiger oxidations. J Mol Catal B Enzym 4:111–136
Sheng D, Ballou DP, Massey V (2001) Mechanistic studies of cyclohexanone monooxygenase: chemical properties of intermediates involved in catalysis. Biochemistry 40:11156–11167
Stewart JD (1998) Cyclohexanone monooxygenase: a useful reagent for asymmetric Baeyer–Villiger reactions. Curr Org Chem 2:211–232
Straathof AJJ, Jongejan JA (1997) The enantiomeric ratio: origin, determination and prediction. Enzyme Microb Technol 21:559–571
Stutzenberger F, Lupo D (1986) pH-dependent thermal activation of endo-1,4-glucanase in Thermomonospora curvata. Enzyme Microb Technol 8:205–208
Vallon O (2000) New sequence motifs in flavoproteins: evidence for common ancestry and tools to predict structure. Proteins 38:95–114
Venter JC, Remington K, Heidelberg JF, Halpern AL, Rusch D, Eisen JA, Wu D, Paulsen I, Nelson KE, Nelson W, Fouts DE, Levy S, Knap AH, Lomas MW, Nealson K, White O, Peterson J, Hoffman J, Parsons R, Baden-Tillson H, Pfannkoch C, Rogers YH, Smith HO (2004) Environmental genome shotgun sequencing of the Sargasso sea. Science 304:66–74
Walton AZ, Stewart JD (2002) An efficient enzymatic Baeyer–Villiger oxidation by engineered Escherichia coli cells under non-growing conditions. Biotechnol Prog 18:262–268
Wilson DB (2004) Studies of Thermobifida fusca plant cell wall degrading enzymes. Chem Rec 4:72–82
Zambianchi F, Pasta P, Carrea G, Colonna S, Gaggero N, Woodley JM (2002) Use of isolated cyclohexanone monooxygenase from recombinant Escherichia coli as a biocatalyst for Baeyer–Villiger and sulfide oxidations. Biotechnol Bioeng 78:489–496
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Fraaije, M.W., Wu, J., Heuts, D.P.H.M. et al. Discovery of a thermostable Baeyer–Villiger monooxygenase by genome mining. Appl Microbiol Biotechnol 66, 393–400 (2005). https://doi.org/10.1007/s00253-004-1749-5
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DOI: https://doi.org/10.1007/s00253-004-1749-5