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Heterologous expression and characterization of Choline Oxidase from the soil bacterium Arthrobacter nicotianae

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Abstract

In the course of a microbial screening of soil samples for new oxidases, different enrichment strategies were carried out. With choline as the only carbon source, a microorganism was isolated and identified as Arthrobacter nicotianae. From this strain, a gene coding for a choline oxidase was isolated from chromosomal DNA. This gene named codA was cloned in Escherichia coli BL21-Gold and the protein (An_CodA) heterologously overexpressed as a soluble intracellular protein of 59.1 kDa. Basic biochemical characterization of purified protein revealed a pH optimum of 7.4 and activity over a broad temperature range (15–70 °C). Specific activities were determined toward choline chloride (4.70 ± 0.12 U/mg) and the synthetic analogs bis(2-hydroxyethyl)-dimethylammonium chloride (0.05 ± 0.45 × 10–2 U/mg) and tris-(2-hydroxyethyl)-methylammonium methylsulfate (0.01 ± 0.12 × 10–2 U/mg). With increasing number of oxidizable groups, a significant decrease in activity was noted. Determination of kinetic parameters in atmorspheric oxygen resulted in K M = 1.51 ± 0.09 mM and V max = 42.73 ± 0.42 mU/min for choline chloride and K M = 4.77 ± 0.76 mM and V max = 48.40 ± 2.88 mU/min for the reaction intermediate betaine aldehyde respectively. Nuclear magnetic resonance spectroscopic analysis of the products formed during the enzyme reaction with choline chloride showed that in vitro the intermediate betaine aldehyde exists also free in solution.

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References

  • Balzer D, Ziegler G, Pansegrau W, Kruft V, Lanka E (1992) KorB protein of promiscuous plasmid RP4 recognizes inverted sequence repetitions essential for conjugative plasmid transfer. Nucleic Acids Res 20:1851–1858

    Article  CAS  Google Scholar 

  • Bentley SD, Brosch R, Gordon SV, Hopwood DA, Cole ST (2004) Genomics of actinobacteria, the high G+C Gram-positive bacteria. Microbial Genomes 2004:333–359

    Google Scholar 

  • Bylina EJ, Coleman WJ, Dilworth MR, Robles SJ, Tanner MA, Yang MM, Youvan DC (2000) Solid phase enzyme screening. ASM News 66:211–217

    Google Scholar 

  • Cavener DR (1992) GMC Oxidoreductases—a newly defiend family of homologous proteins with diverse catalytic activities. J Mol Biol 223:811–814

    Article  CAS  Google Scholar 

  • Delgrave S, Murphy DJ, Rittenhouse pruss JL, Maffia AM, Marrs BL, Bylina EJ, Coleman WJ, Grek CL, Dilworth MR, Yang MM, Youvan DC (2001) Application of a very high-throughput digital imaging screen to evolve the enzyme galactose oxidase. Protein Eng 14(4):261–267

    Article  Google Scholar 

  • Dym O, Eisenberg D (2001) Sequence structure analysis of FAD-containing proteins. Protein Sci 10:1712–1728

    Article  CAS  Google Scholar 

  • Fan F, Gadda G (2005) On the catalytic mechanism of choline oxidase. J Am Soc 127:2067–2074

    Article  CAS  Google Scholar 

  • Fan F, Ghanem M, Gadda G (2004) Cloning, sequence analysis, and purification of choline oxidase from Arthrobacter globiformis: a bacterial enzyme involved in osmotic stress tolerance. Arch Biochem Biophys 421:149–158

    Article  CAS  Google Scholar 

  • Ghanem M, Gadda G (2005) On the catalytic role of the conserved active site residue His466 of choline oxidase. Biochemistry 44:893–904

    Article  CAS  Google Scholar 

  • Hatefi Y, Stigall DL (1976) Metal-containing flavoprotein dehydrogenase, 3rd Edn. Enzymes 13:175–297

    Article  CAS  Google Scholar 

  • Kawahara Y, Yoshihara Y, Ikeda S, Hirose Y (1990) Effect of glycine betaine, an osmoprotective compound on the growth of Brevibacterium lactofermentum. Appl Microbiol Biotechnol 33(5):574–7

    Article  CAS  Google Scholar 

  • Kempf B, Bremer E (1998) Uptake and synthesis of compatible solutes as microbial stress responses to high-osmolality environments. Arch Microbiol 170:319–330

    Article  CAS  Google Scholar 

  • Kiess M, Hecht HJ, Kalisz HM (1998) Glucose oxidase from Penicillium amagasakiense—primary structure and comparison with other glucose-methanol-choline-(GMC) oxidoreductases. Eur J Biochem 252:90–99

    Article  CAS  Google Scholar 

  • Kunte HJ (2006) Osmoregulation in bacteria: compatible solute accumulation and osmosensing. Environ Chem 3:94–99

    Article  CAS  Google Scholar 

  • Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685

    Article  CAS  Google Scholar 

  • Landfald B, Strom AR (1986) Choline—glycine betaine pathway confers a high level of osmotic tolerance in Escherichia coli. J Bacteriol 165:849–855

    Article  CAS  Google Scholar 

  • Macheroux P, Fitzpatrick TB, Amrhein N (2003) Characterization of YqjM, an Old Yellow Enzyme Homolog from Bacillus subtilis Involved in the Oxidative Stress Response. J Biol Chem 278(22):19891–19897

    Article  Google Scholar 

  • Ohta M, Miura R, Yamano T, Miyake Y (1983) Spectroscopic studies on the photoreaction of choline oxidase, a flavoprotein, with covalently bound flavin. J Biochem 94:879–892

    Article  CAS  Google Scholar 

  • Quaye O, Lountos GT, Fan F, Orville AM, Gadda G (2008) Role of Glu312 in Binding and Positioning of the Substrate for the Hydride Transfer Reaction in Choline Oxidase. Biochemistry 47(1):243–256

    Article  CAS  Google Scholar 

  • Rathinasabapathi B, Burnet M, Russell BL, Gage DA, Liao P, Nye GJ, Scott P, Golbeck JH, Hanson AD (1997) Choline monooxygenase, an unusual iron-sulfur enzyme catalyzing the first step of glycine betaine synthesis in plants: Prosthetic group, characterization and cDNA cloning. Proc Natl Acad Sci USA 94(7):3454–3458

    Article  CAS  Google Scholar 

  • Rhodes D, Hanson AD (1993) Quaternary ammonium and tertiary sulfonium compounds in higher plants. Annu Rev Plant Phyiol Plant Mol Biol 44:357–384

    Article  CAS  Google Scholar 

  • Rozwadowski KL, Khachatourians GG, Selvaraj G (1991) Choline oxidase, a catabolic enzyme in Arthrobacter pascens, facilitates adaptation to osmotic stress in Escherichia coli. J Bacteriol 173(2):472–478

    Article  CAS  Google Scholar 

  • Rungsrisuriyachai K, Gadda G (2008) On the role of histidine 351 in the reaction of alcohol oxidation catalyzed by choline oxidase. Biochemistry 47(26):6762–6769

    Article  CAS  Google Scholar 

  • Sakamoto A, Murata N (2001) The use of bacterial choline oxidase, a glycinebetaine-synthesizing enzyme, to create stress-resistant transgenic plants. Plant Physiol 125:180–188

    Article  CAS  Google Scholar 

  • Sakamoto A, Murata A, Murata N (1998) Metabolic engineering of rice leading to biosynthesis of glycinebetaine and tolerance to salt and cold. Plant Mol Biol 38:1011–1019

    Article  CAS  Google Scholar 

  • Sakamoto A, Valverde R, Murata A, Chen T, Murata N (2000) Transformation of Arabidopsis with the codA gene for choline oxidase enhances freezing tolerance of plants. Plant J 22(5):449–453

    Article  CAS  Google Scholar 

  • Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a labatory manual, 2nd edn. Cold Spring Harbor Laboratory Press, Cold Sprig Harbor

    Google Scholar 

  • Sanger F, Nicklen S, Coulson AR (1977) DNA sequencing with chain-terminating inhibitors. PNAS 74(12):5463–5467

    Article  CAS  Google Scholar 

  • Schlegel HG (1985) Allgemeine Microbiologie. Georg Thieme Verlag Stuttgart, New York

    Google Scholar 

  • Weretilnyk EA, Hanson AD (1990) Molecular cloning of a plant betaine - aldehyde dehydrogenase, an enzyme implicated in adaptation to salinity and drought. Proc Natl Acad Sci USA 87(7):2745–49

    Article  CAS  Google Scholar 

  • Wierenga RK, Terpstra P, Hol WG (1986) Prediction of the occurrence of the ADP- binding bab-fold in proteins, using an amino acid sequence fingerprint. J Mol Biol 187:101–107

    Article  CAS  Google Scholar 

  • Wong RC, Ngo TT, Lenhoff HM (1981) Formation of blue chromophore from oxidative coupling of aminoantipyrine with chromotopic acid in the presence of peroxide and horseradish peroxidase. Int J Biochem 13:159–163

    Article  CAS  Google Scholar 

Download references

Acknowledgment

This study was performed within the Competence Centre “Applied Biocatalysis” in cooperation with Henkel AG and Co. KGaA. Financial support from the FFG, the City of Graz, and the Province of Styria is gratefully acknowledged.

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Authors and Affiliations

  1. Research Centre Applied Biocatalysis, Petersgasse 14, 8010, Graz, Austria

    D. Ribitsch, W. Karl, E. Wehrschütz-Sigl, S. Tutz, P. Remler, K. Gruber & H. Schwab

  2. Institute of Molecular Biotechnology, Graz University of Technology, Petersgasse 14, 8010, Graz, Austria

    H. Schwab

  3. Institute of Organic Chemistry, Graz University of Technology, Stremayrgasse 16, 8010, Graz, Austria

    H. J. Weber

  4. Institute of Molecular Biosciences, Karl-Franzens-University Graz, Humboldtstrasse 50/3, 8010, Graz, Austria

    K. Gruber

  5. Henkel AG and Co. KGaA, Henkelstrasse 67, 40191, Duesseldorf, Germany

    R. Stehr, C. Bessler, N. Hoven, K. Sauter & K. H. Maurer

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  1. D. Ribitsch
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Correspondence to H. Schwab.

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Ribitsch, D., Karl, W., Wehrschütz-Sigl, E. et al. Heterologous expression and characterization of Choline Oxidase from the soil bacterium Arthrobacter nicotianae . Appl Microbiol Biotechnol 81, 875–886 (2009). https://doi.org/10.1007/s00253-008-1661-5

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  • DOI: https://doi.org/10.1007/s00253-008-1661-5

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