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Protein engineering of microbial cholesterol oxidases: a molecular approach toward development of new enzymes with new properties

Applied Microbiology and Biotechnology volume 100, pages 4323–4336 (2016)Cite this article

Abstract

Cholesterol oxidase, a flavoenzyme, catalyzes two reactions in one active site: oxidation and isomerization. This enzyme has been isolated from a variety of microorganisms, mostly from actinomycetes. This enzyme has been widely used in clinical laboratories for cholesterol assays and was subsequently determined to have other potential applications. Engineering of cholesterol oxidase have enabled the identification of critical residues, and the information derived could lead to the rational development of improved types of the enzyme with increased stability and better functional properties. This review is the first that exclusively summarizes the reported results on the engineering of bacterial cholesterol oxidases aimed at improving their thermal and chemical stability, catalytic activity, and substrate specificity.

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References

  • Ahire JJ, Bhat AA, Thakare JM, Pawar PB, Zope DG, Jain RM, Chaudhari BL (2012) Cholesterol assimilation and biotransformation by Lactobacillus helveticus. Biotechnol Lett 34(1):103–107

    CAS  Article  PubMed  Google Scholar 

  • Allain CC, Poon LS, Chan CS, Richmond W, Fu PC (1974) Enzymatic determination of total serum cholesterol. Clin Chem 20(4):470–475

    CAS  PubMed  Google Scholar 

  • Aparicio JF, Martin JF (2008) Microbial cholesterol oxidases: bioconversion enzymes or signal proteins? Mol BioSyst 4(8):804–809

    CAS  Article  PubMed  Google Scholar 

  • Caldinelli L, Iametti S, Barbiroli A, Bonomi F, Fessas D, Molla G, Pilone MS, Pollegioni L (2005) Dissecting the structural determinants of the stability of cholesterol oxidase containing covalently bound flavin. J Biol Chem 280(24):22572–22581

    CAS  Article  PubMed  Google Scholar 

  • Coulombe R, Yue KQ, Ghisla S, Vrielink A (2001) Oxygen access to the active site of cholesterol oxidase through a narrow channel is gated by an Arg-Glu pair. J Biol Chem 276(32):30435–30441

  • de las Heras LF, Perera J, Llorens JMN (2014) Cholesterol to cholestenone oxidation by ChoG, the main extracellular cholesterol oxidase of Rhodococcus ruber strain Chol-4. J Steroid Biochem Mol Biol 139:33–44

    Article  Google Scholar 

  • Dijkman WP, de Gonzalo G, Mattevi A, Fraaije MW (2013) Flavoprotein oxidases: classification and applications. Appl Microbiol Biotechnol 97(12):5177–5188

    CAS  Article  PubMed  Google Scholar 

  • Doukyu N (2009) Characteristics and biotechnological applications of microbial cholesterol oxidases. Appl Microbiol Biotechnol 83(5):825–837

    CAS  Article  PubMed  Google Scholar 

  • Doukyu N, Aono R (2001) Cloning, sequence analysis and expression of a gene encoding an organic solvent- and detergent-tolerant cholesterol oxidase of Burkholderia cepacia strain ST-200. Appl Microbiol Biotechnol 57(1–2):146–152

    CAS  PubMed  Google Scholar 

  • Doukyu N, Shibata K, Ogino H, Sagermann M (2008) Purification and characterization of Chromobacterium sp. DS-1 cholesterol oxidase with thermal, organic solvent, and detergent tolerance. Appl Microbiol Biotechnol 80(1):59–70

    CAS  Article  PubMed  Google Scholar 

  • Doukyu N, Shibata K, Ogino H, Sagermann M (2009) Cloning, sequence analysis, and expression of a gene encoding Chromobacterium sp. DS-1 cholesterol oxidase. Appl Microbiol Biotechnol 82(3):479–490

    CAS  Article  PubMed  Google Scholar 

  • Fujishiro K, Uchida H, Shimokawa K, Nakano M, Sano F, Ohta T, Kayahara N, Aisaka K, Uwajima T (2002) Purification and properties of a new Brevibacterium sterolicum cholesterol oxidase produced by E. coli MM294/pnH10. FEMS Microbiol Lett 215(2):243–248

    CAS  Article  PubMed  Google Scholar 

  • Gamba P, Leonarduzzi G, Tamagno E, Guglielmotto M, Testa G, Sottero B, Gargiulo S, Biasi F, Mauro A, Vina J (2011) Interaction between 24-hydroxycholesterol, oxidative stress, and amyloid-β in amplifying neuronal damage in Alzheimer’s disease: three partners in crime. Aging Cell 10(3):403–417

    CAS  Article  PubMed  Google Scholar 

  • Ghasemian A, Yazdi MH, Sepehrizadeh Z (2008) Construction of a thermally stable cholesterol oxidase mutant by site-directed mutagenesis. Biotechnology 7:826–829

    CAS  Article  Google Scholar 

  • Ghasemian A, Yazdi M, Sepehrizadeh Z, Yazdi Z, Zarrini G (2009) Overexpression, one-step purification, and characterization of a type II cholesterol oxidase from a local isolate Rhodococcus sp. PTCC 1633. World J Microb Biot 25(5):773–779

    CAS  Article  Google Scholar 

  • Goswami P, Chinnadayyala SSR, Chakraborty M, Kumar AK, Kakoti A (2013) An overview on alcohol oxidases and their potential applications. Appl Microbiol Biotechnol 97(10):4259–4275

    CAS  Article  PubMed  Google Scholar 

  • Javid Khalili S, Sepehrizadeh Z, Tabatabaei Yazdi M, Ghasemian A, Zargar S (2009) Study of the importance of Glu361 in the active site of cholesterol oxidase from Rhodococcus sp. PTCC 1633 by site-directed mutagenesis. Ann Microbiol 59(2):395–397

    Article  Google Scholar 

  • Kass IJ, Sampson NS (1998) The importance of GLU361 position in the reaction catalyzed by cholesterol oxidase. Bioorg Med Chem Lett 8(19):2663–2668

    CAS  Article  PubMed  Google Scholar 

  • Khan R, Solanki P, Kaushik A, Singh S, Ahmad S, Malhotra B (2009) Cholesterol biosensor based on electrochemically prepared polyaniline conducting polymer film in presence of a nonionic surfactant. J Polym Res 16(4):363–373

    CAS  Article  Google Scholar 

  • Kojima K, Kobayashi T, Tsugawa W, Ferri S, Sode K (2013) Mutational analysis of the oxygen-binding site of cholesterol oxidase and its impact on dye-mediated dehydrogenase activity. J Mol Catal B Enzym 88:41–46

    CAS  Article  Google Scholar 

  • Kreit J, Sampson NS (2009) Cholesterol oxidase: physiological functions. FEBS J 276(23):6844–6856

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  • Kumari L, Kanwar SS (2012) Cholesterol oxidase and its applications. Adv Microbiol 2(2):49–65

    CAS  Article  Google Scholar 

  • Lario PI, Sampson N, Vrielink A (2003) Sub-atomic resolution crystal structure of cholesterol oxidase: what atomic resolution crystallography reveals about enzyme mechanism and the role of the FAD cofactor in redox activity. J Mol Biol 326(5):1635–1650

    CAS  Article  PubMed  Google Scholar 

  • Lashkarian H, Raheb J, Shahzamani K, Shahbani H, Shamsara M (2010) Extracellular cholesterol oxidase from Rhodococcus sp.: isolation and molecular characterization. Iran Biomed J 14(1–2):49

    CAS  PubMed  PubMed Central  Google Scholar 

  • Li J, Vrielink A, Brick P, Blow DM (1993) Crystal structure of cholesterol oxidase complexed with a steroid substrate: implications for flavin adenine dinucleotide dependent alcohol oxidases. Biochemistry 32(43):11507–11515

    CAS  Article  PubMed  Google Scholar 

  • Lim L, Molla G, Guinn N, Ghisla S, Pollegioni L, Vrielink A (2006) Structural and kinetic analyses of the H121A mutant of cholesterol oxidase. Biochem J 400(1):13–22

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  • Lin Y, Fu J, Song X (2010) Purification and characterization of an extracellular cholesterol oxidase from a Bordetella species. Process Biochem 45(9):1563–1569

    CAS  Article  Google Scholar 

  • MacLachlan J, Wotherspoon AT, Ansell RO, Brooks CJ (2000) Cholesterol oxidase: sources, physical properties and analytical applications. J Steroid Biochem Mol Biol 72(5):169–195

    CAS  Article  PubMed  Google Scholar 

  • Mendes MV, Recio E, Antón N, Guerra SM, Santos-Aberturas J, Martín JF, Aparicio JF (2007) Cholesterol oxidases act as signaling proteins for the biosynthesis of the polyene macrolide pimaricin. Chem Biol 14(3):279–290

    CAS  Article  PubMed  Google Scholar 

  • Molaei R, Sabzi RE, Farhadi K, Kheiri F, Forough M (2014) Amperometric biosensor for cholesterol based on novel nanocomposite array gold nanoparticles/acetone-extracted propolis/multiwall carbon nanotubes/gold. Micro Nano Lett 9(2):100–104

    Article  Google Scholar 

  • Molnár I, Choi K-P, Hayashi N, Murooka Y (1991) Secretory overproduction of Streptomyces cholesterol oxidase by Streptomyces lividans with a multi-copy shuttle vector. J Ferment Bioeng 72(5):368–372

    Article  Google Scholar 

  • Motteran L, Pilone MS, Molla G, Ghisla S, Pollegioni L (2001) Cholesterol oxidase from Brevibacterium sterolicum. The relationship between covalent flavinylation and redox properties. J Biol Chem 276(21):18024–18030

    CAS  Article  PubMed  Google Scholar 

  • Murooka Y, Ishizaki T, Nimi O, Maekawa N (1986) Cloning and expression of a Streptomyces cholesterol oxidase gene in Streptomyces lividans with plasmid pIJ702. Appl Environ Microbiol 52(6):1382–1385

    CAS  PubMed  PubMed Central  Google Scholar 

  • Navas J, Gonzalez-Zorn B, Ladron N, Garrido P, Vazquez-Boland JA (2001) Identification and mutagenesis by allelic exchange of choE, encoding a cholesterol oxidase from the intracellular pathogen Rhodococcus equi. J Bacteriol 183(16):4796–4805

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  • Nishiya Y, Hirayama N (1999) Alteration of substrate affinity of Streptomyces cholesterol oxidase for application to the rate assay of cholesterol in serum. Clin Chim Acta 287(1–2):111–122

    CAS  Article  PubMed  Google Scholar 

  • Nishiya Y, Harada N, Teshima SI, Yamashita M, Fujii I, Hirayama N, Murooka Y (1997) Improvement of thermal stability of Streptomyces cholesterol oxidase by random mutagenesis and a structural interpretation. Protein Eng 10(3):231–235

    CAS  Article  PubMed  Google Scholar 

  • Nishiya Y, Yamashita M, Murooka Y, Fujii I, Hirayama N (1998) Effect of non-ionic detergents on apparent enzyme mechanism: V121A mutant of Streptomyces cholesterol oxidase endowed with enhanced sensitivity towards detergents. Protein Eng 11(8):609–611

    CAS  Article  PubMed  Google Scholar 

  • Nomura N, Choi K, Yamashita M, Yamamoto H, Murooka Y (1995) Genetic modification of the Streptomyces cholesterol oxidase gene for expression in Escherichia coli and development of promoter-probe vectors for use in enteric bacteria. J Ferment Bioeng 79(5):410–416

    CAS  Article  Google Scholar 

  • Ohta T, Fujishiro K, Yamaguchi K, Uwajima T, Aisaka K, Hasegawa M (1992) Hyperexpression and analysis of choB encoding cholesterol oxidase of Brevibacterium sterolicum in Escherichia coli and Streptomyces lividans. Biosci Biotechnol Biochem 56(11):1786–1791

    CAS  Article  PubMed  Google Scholar 

  • Piubelli L, Pedotti M, Molla G, Feindler-Boeckh S, Ghisla S, Pilone MS, Pollegioni L (2008) On the oxygen reactivity of flavoprotein oxidases an oxygen access tunnel and gate in Brevibacterium sterolicum cholesterol oxidase. J Biol Chem 283(36):24738–24747

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  • Pollegioni L (2009) Cholesterol oxidase: a model flavoprotein oxidase and a biotechnological tool. FEBS J 276(23):6825–6825

    CAS  Article  PubMed  Google Scholar 

  • Pollegioni L, Piubelli L, Molla G (2009) Cholesterol oxidase: biotechnological applications. FEBS J 276(23):6857–6870

    CAS  Article  PubMed  Google Scholar 

  • Puglielli L, Friedlich AL, Setchell KD, Nagano S, Opazo C, Cherny RA, Barnham KJ, Wade JD, Melov S, Kovacs DM (2005) Alzheimer disease β-amyloid activity mimics cholesterol oxidase. J Clin Invest 115(9):2556–2563

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  • Purcell JP, Greenplate JT, Jennings MG, Ryerse JS, Pershing JC, Sims SR, Prinsen MJ, Corbin DR, Tran M, Sammons RD, Stonard RJ (1993) Cholesterol oxidase: a potent insecticidal protein active against boll weevil larvae. Biochem Biophys Res Commun 196(3):1406–1413

    CAS  Article  PubMed  Google Scholar 

  • Sampson NS, Chen X (1998) Increased expression of Brevibacterium sterolicum cholesterol oxidase in Escherichia coli by genetic modification. Protein Expr Purif 12(3):347–352

    CAS  Article  PubMed  Google Scholar 

  • Sampson NS, Vrielink A (2003) Cholesterol oxidases: a study of nature’s approach to protein design. Acc Chem Res 36(9):713–722

    CAS  Article  PubMed  Google Scholar 

  • Sojo M, Bru R, Lopez-Molina D, Garcia-Carmona F, Arguelles JC (1997) Cell-linked and extracellular cholesterol oxidase activities from Rhodococcus erythropolis. Isolation and physiological characterization. Appl Microbiol Biotechnol 47(5):583–589

    CAS  Article  PubMed  Google Scholar 

  • Sottero B, Gamba P, Gargiulo S, Leonarduzzi G, Poli G (2009) Cholesterol oxidation products and disease: an emerging topic of interest in medicinal chemistry. Curr Med Chem 16(6):685–705

    CAS  Article  PubMed  Google Scholar 

  • Sun Y, Yang H, Wang W (2011) Improvement of the thermostability and enzymatic activity of cholesterol oxidase by site-directed mutagenesis. Biotechnol Lett 33(10):2049–2055

    CAS  Article  PubMed  Google Scholar 

  • Toyama M, Yamashita M, Yoneda M, Zaborowski A, Nagato M, Ono H, Hirayama N, Murooka Y (2002) Alteration of substrate specificity of cholesterol oxidase from Streptomyces sp. by site-directed mutagenesis. Protein Eng 15(6):477–484

    CAS  Article  PubMed  Google Scholar 

  • Uwajima T, Yagi H, Terada O (1974) Properties of crystalline 3β-hydroxysteroid oxidase of Brevibacterium sterolicam. Agric Biol Chem 38:1149–1156

    CAS  Article  Google Scholar 

  • Volonte F, Pollegioni L, Molla G, Frattini L, Marinelli F, Piubelli L (2010) Production of recombinant cholesterol oxidase containing covalently bound FAD in Escherichia coli. BMC Biotechnol 10:33

    Article  PubMed  PubMed Central  Google Scholar 

  • Vrielink A, Ghisla S (2009) Cholesterol oxidase: biochemistry and structural features. FEBS J 276(23):6826–6843

    CAS  Article  PubMed  Google Scholar 

  • Vrielnik A (2010) Cholesterol oxidase: structure and function. Cholesterol binding and cholesterol transport proteins. Springer Sciences + Business Media B.V. Subcell Biochem 51:137–158

    Article  Google Scholar 

  • Wang L, Wang W (2007) Coenzyme precursor-assisted expression of a cholesterol oxidase from Brevibacterium sp. in Escherichia coli. Biotechnol Lett 29(5):761–766

    CAS  Article  PubMed  Google Scholar 

  • Xiang J, Sampson NS (2004) Library screening studies to investigate substrate specificity in the reaction catalyzed by cholesterol oxidase. Protein Eng Des Sel 17(4):341–348

    CAS  Article  PubMed  Google Scholar 

  • Yamashita M, Toyama M, Ono H, Fujii I, Hirayama N, Murooka Y (1998) Separation of the two reactions, oxidation and isomerization, catalyzed by Streptomyces cholesterol oxidase. Protein Eng 11(11):1075–1081

    CAS  Article  PubMed  Google Scholar 

  • Yao K, Wang F-Q, Zhang H-C, Wei D-Z (2013) Identification and engineering of cholesterol oxidases involved in the initial step of sterols catabolism in Mycobacterium neoaurum. Metab Eng 15:75–87

    CAS  Article  PubMed  Google Scholar 

  • Yazdi MT, Yazdi ZT, Ghasemian A, Zarrmi G, Olyaee NH, Sepehrizadeh Z (2008) Purification and characterization of extra-cellular cholesterol oxidase from Rhodococcus sp. PTCC 1633. Biotechnology 7(4):751–756

    CAS  Article  Google Scholar 

  • Yin Y, Liu P, Anderson RG, Sampson NS (2002) Construction of a catalytically inactive cholesterol oxidase mutant: investigation of the interplay between active site-residues glutamate 361 and histidine 447. Arch Biochem Biophys 402(2):235–242

    CAS  Article  PubMed  Google Scholar 

  • Yue QK, Kass IJ, Sampson NS, Vrielink A (1999) Crystal structure determination of cholesterol oxidase from Streptomyces and structural characterization of key active site mutants. Biochemistry 38(14):4277–4286

    CAS  Article  PubMed  Google Scholar 

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  1. Department of Pharmaceutical Biotechnology, Faculty of Pharmacy, Urmia University of Medical Sciences, P.O. Box 57157-1441, Urmia, Iran

    Zahra Moradpour & Abdollah Ghasemian

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  1. Zahra Moradpour
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  2. Abdollah Ghasemian
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Correspondence to Abdollah Ghasemian.

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The authors have contributed sufficiently to the work and therefore share collective responsibility for the manuscript. This article does not contain any studies with human participants or animals. All authors have approved the manuscript.

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Moradpour, Z., Ghasemian, A. Protein engineering of microbial cholesterol oxidases: a molecular approach toward development of new enzymes with new properties. Appl Microbiol Biotechnol 100, 4323–4336 (2016). https://doi.org/10.1007/s00253-016-7497-5

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  • DOI: https://doi.org/10.1007/s00253-016-7497-5

Keywords

  • Cholesterol oxidase
  • Flavoenzyme
  • Protein engineering
  • Site-directed mutagenesis
  • Thermostable enzyme