Browse > Article
http://dx.doi.org/10.4014/jmb.1911.11048

Characterization of Two Self-Sufficient Monooxygenases, CYP102A15 and CYP102A170, as Long-Chain Fatty Acid Hydroxylases  

Rimal, Hemraj (Department of Life Science and Biochemical Engineering, Sunmoon University)
Lee, Woo-Haeng (Department of Life Science and Biochemical Engineering, Sunmoon University)
Kim, Ki-Hwa (Department of Life Science and Biochemical Engineering, Sunmoon University)
Park, Hyun (Division of Biotechnology, College of Life Sciences and Biotechnology, Korea University)
Oh, Tae-Jin (Department of Life Science and Biochemical Engineering, Sunmoon University)
Publication Information
Journal of Microbiology and Biotechnology / v.30, no.5, 2020 , pp. 777-784 More about this Journal
Abstract
Self-sufficient P450s, due to their fused nature, are the most effective tools for electron transfer to activate C-H bonds. They catalyze the oxygenation of fatty acids at different omega positions. Here, two new, self-sufficient cytochrome P450s, named 'CYP102A15 and CYP102A170,' from polar Bacillus sp. PAMC 25034 and Paenibacillus sp. PAMC 22724,respectively, were cloned and expressed in E. coli. The genes are homologues of CYP102A1 from Bacillus megaterium. They catalyzed the hydroxylation of both saturated and unsaturated fatty acids ranging in length from C12-C20, with a moderately diverse profile compared to other members of the CYP102A subfamily. CYP102A15 exhibited the highest activity toward linoleic acid with Km 15.3 μM, and CYP102A170 showed higher activity toward myristic acid with Km 17.4 μM. CYP10A170 also hydroxylated the Eicosapentaenoic acid at ω-1 position only. Various kinetic parameters of both monooxygenases were also determined.
Keywords
Bacillus sp.; cytochrome P450; fatty acid hydroxylation; Paenibacillus sp.; self-sufficient monooxygenase;
Citations & Related Records
연도 인용수 순위
  • Reference
1 Khatri Y, Hannemann F, Girhard M, Kappl R, Hutter M, Urlacher VB, et al. 2015. A natural heme-signature variant of CYP267A1 from Sorangium cellulosum So ce56 executes diverse $\omega$-hydroxylation. FEBS J. 282: 74-88.   DOI
2 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.   DOI
3 Bhattarai S, Liou K, Oh TJ. 2013. Hydroxylation of long chain fatty acids by CYP147F1, a new cytochrome P450 subfamily protein from Streptomyces peucetius. Arch. Biochem. Biophys. 539: 63-69.   DOI
4 Khatri Y, Hannemann F, Ewen KM, Pistorius D, Perlova O, Kagawa N, et al. 2010. The CYPome of Sorangium cellulosum So ce56 and identification of CYP109D1 as a new fatty acid hydroxylase. Chem. Biol. 17: 1295-1305.   DOI
5 Budde M, Maurer SC, Schmid RD, Urlacher VB. 2004. Cloning, expression and characterisation of CYP102A2, a self-sufficient P450 monooxygenase from Bacillus subtilis. Appl. Microbiol. Biotechnol. 66: 180-186.   DOI
6 Lentz O, Urlacher V, Schmid RD. 2004. Substrate specificity of native and mutated cytochrome P450 (CYP102A3) from Bacillus subtilis. J. Biotechnol. 108: 41-49.   DOI
7 Porter JL, Manning J, Sabatini S, Tavanti M, Turner NJ, Flitsch SL. 2018. Characterisation of CYP102A25 from Bacillus marmarensis and CYP102A26 from Pontibacillus halophilus?: P450 homologues of BM3 with preference towards hydroxylation of medium-chain fatty acids. ChembioChem 19: 513-520.   DOI
8 Dietrich M, Eiben S, Asta C, Do TA, Pleiss J, Urlacher VB. 2008. Cloning, expression and characterisation of CYP102A7, a selfsufficient P450 monooxygenase from Bacillus licheniformis. Appl. Microbiol. Biotechnol. 79: 931-940.   DOI
9 Fulco AJ. 1991. P450BM-3 and other inducible bacterial P450 cytochromes: biochemistry and regulation. Annu. Rev. Pharmacol. Toxicol. 31: 177-203.   DOI
10 Palmer CN, Axen E, Hughes V, Wolf CR. 1998. The repressor protein, Bm3R1, mediates an adaptive response to toxic fatty acids in Bacillus megaterium. J. Biol. Chem. 273: 18109-18116.   DOI
11 Gustafsson MCU, Roitel O, Marshall KR, Noble MA, Chapman SK, Pessequeiro A, et al. 2004. Expression, purification, and characterization of Bacillus subtilis cytochromes P450 CYP102A2 and CYP102A3: flavocytochrome homologues of P450 BM3 from Bacillus megaterium. Biochemistry 43: 5474-5487.   DOI
12 Chung YH, Song JW, Choi KY, Yoon JW, Yang KM, Park JB. 2012. Cloning, expression, and characterization of P450 monooxygenase CYP102H1 from Nocardia farcinica. J. Korean Soc. Appl. Biol. Chem. 55: 259-264.   DOI
13 Munday SD, Maddigan NK, Young RJ, Bell SG. 2016. Characterisation of two self-sufficient CYP102 family monooxygenases from Ktedonobacter racemifer DSM44963 which have new fatty acid alcohol product profiles. Biochim. Biophys. Acta 1860: 1149-1162.   DOI
14 Choi KY, Jung E, Jung DH, Pandey BP, Yun H, Park HY, et al. 2012. Cloning, expression and characterization of CYP102D1, a selfsufficient P450 monooxygenase from Streptomyces avermitilis. FEBS J. 279: 1650-1662.   DOI
15 Lamb DC, Lei L, Zhao B, Yuan H, Jackson CJ, Warrilow AG, et al. 2010. Streptomyces coelicolor A3(2) CYP102 protein, a novel fatty acid hydroxylase encoded as a heme domain without an N-terminal redox partner. Appl. Environ. Microbiol. 76: 1975-1980.   DOI
16 Narhi LO, Fulco AJ. 1982. Phenobarbital induction of a soluble cytochrome P-450-dependent fatty acid monooxygenase in Bacillus megaterium. J. Biol. Chem. 257: 2147-2150.   DOI
17 Kimura M. 1980. A simple method for estimating evolutionary rates of base substitutions through comparative studies of nucleotide sequences. J. Mol. Evol. 16: 111-120.   DOI
18 Chowdhary PK, Alemseghed M, Haines DC. 2007. Cloning, expression and characterization of a fast self-sufficient P450: CYP102A5 from Bacillus cereus. Arch. Biochem. Biophys. 468: 32-43.   DOI
19 Nestl BM, Hammer SC, Nebel BA, Hauer B. 2014. New generation of biocatalysts for organic synthesis. Angew Chemie. Int. Ed. 53: 3070-3095.   DOI
20 Tamura K, Stecher G, Peterson D, Filipski A, Kumar S. 2013. MEGA6: Molecular evolutionary genetics analysis version 6.0. Mol. Biol. Evol. 30: 2725-2729.   DOI
21 Saitou N, Nei M. 1987. The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol. Biol. Evol. 4: 406-425.
22 Minerdi D, Sadeghi SJ, Di Nardo G, Rua F, Castrignano S, Allegra P, et al. 2015. CYP116B5: a new class VII catalytically self-sufficient cytochrome P450 from Acinetobacter radioresistens that enables growth on alkanes. Mol. Microbiol. 95: 539-554.   DOI
23 Gunsalus IC, Sligar SG. 1978. Oxygen reduction by the P450 monoxygenase systems. Adv. Enzymol. Relat. Areas Mol. Biol. 47: 1-44.
24 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.   DOI
25 Aliverti A, Curti B, Vanoni MA. 1999. Identifying and quantitating FAD and FMN in simple and in iron-sulfur-containing flavoproteins. pp. 9-24. In: flavoprotein protocols. Humana Press, New Jersey.
26 Shareef A, Angove MJ, Wells JD. 2006. Optimization of silylation using N-methyl-N-(trimethylsilyl)-trifluoroacetamide, N,O-bis-(trimethylsilyl)-trifluoroacetamide and N-(tert-butyldimethylsilyl)-N-methyltrifluoroacetamide for the determination of the estrogens estrone and 17alpha-ethinylestradiol by gas chromatography-mass spectrometry. J. Chromatogr. A 1108: 121-128.   DOI
27 Urlacher VB, Eiben S. 2006. Cytochrome P450 monooxygenases: perspectives for synthetic application. Trends Biotechnol. 24: 324-330.   DOI
28 Werck-Reichhart D, Feyereisen R. 2000. Cytochromes P450: a success story. Genome Biol. 1: Reviews3003.
29 McLean KJ, Sabri M, Marshall KR, Lawson RJ, Lewis DG, Clift D, et al. 2005. Biodiversity of cytochrome P450 redox systems. Biochem. Soc. Trans. 33: 796-801.   DOI
30 Schmid A, Hollmann F, Park JB, Buhler B. 2002. The use of enzymes in the chemical industry in Europe. Curr. Opin. Biotechnol. 13: 359-366.   DOI
31 Boddupalli SS, Estabrook RW, Peterson JA. 1990. Fatty acid monooxygenation by cytochrome P-450BM-3. J. Biol. Chem. 265: 4233-4239.   DOI
32 Truan G, Komandla MR, Falck JR, Peterson JA. 1999. P450BM-3: absolute configuration of the primary metabolites of palmitic acid. Arch. Biochem. Biophys. 366: 192-198.   DOI
33 Miura Y, Fulco AJ. 1975. Omega-1, Omega-2 and Omega-3 hydroxylation of long-chain fatty acids, amides and alcohols by a soluble enzyme system from Bacillus megaterium. Biochim. Biophys. Acta 388: 305-317.   DOI
34 Budde M, Morr M, Schmid RD, Urlacher VB. 2006. Selective hydroxylation of highly branched fatty acids and their derivatives by CYP102A1 from Bacillus megaterium. ChemBiochem 7: 789-794.   DOI
35 Cryle MJ, Espinoza RD, Smith SJ, Matovic NJ, De Voss JJ. 2006. Are branched chain fatty acids the natural substrates for P450(BM3)? Chem. Commun. (Camb) 14: 2353-2355.
36 Haines DC, Tomchick DR, Machius M, Peterson JA. 2001. Pivotal role of water in the mechanism of P450BM-3. Biochemistry 40: 13456-13465.   DOI
37 English N, Hughes V, Wolf CR. 1994. Common pathways of cytochrome P450 gene regulation by peroxisome proliferators and barbiturates in Bacillus megaterium ATCC14581. J. Biol. Chem. 269: 26836-26841.   DOI