Abstract
A protein with a mol. mass of 51,000 (ThcE) that was induced in Rhodococcus sp. N186/21 during assimilation of thiocarbamate herbicides, atrazine, ethanol, propanol, glycerol, propionaldehyde or ethanolamine was identified by two-dimensional electrophoresis. The thcE gene was cloned and sequenced. The deduced amino acid sequence revealed ThcE as a member of group III alcohol dehydrogenases. ThcE displayed strong homology with sequenced subunit fragments of the homodecameric N,N′-dimethyl-4-nitrosoaniline-dependent alcohol oxidoreductases (MNO) of Amycolatopsis methanolica and Mycobacterium gastri. N-Terminal sequence analysis of purified MNO from Rhodococcus sp. NI86/21 confirmed the identity with ThcE. When overproduced in Escherichia coli, ThcE was insoluble and no MNO activity was detected.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
Abbreviations
- BSM :
-
Basal salt medium
- EPTC :
-
S-ethyl dipropylthiocarbamate
- MDH :
-
methanol dehydrogenase
- MNO :
-
methanol
- NDMA:
-
oxidoreductase
- NDMA :
-
N,N′-dimethyl-4-nitrosoaniline
References
Altschul SF, Gish W, Miller W, Myersand EW, Lipman DJ (1990) Basic local alignment search tool. J Mol Biol 215:403–410
Arfman N, Watling EM, Clement W, Van Oosterwijk RJ, De Vries GE, Harder W, Attwood MM, Dijkhuizen L (1989) Methanol metabolism in thermotolerant methylotrophic Bacillus strains involving a novel catabolic NAD-dependent methanol dehydrogenase as a key enzyme. Arch Microbiol 152:280–288
Arfman N, Van Beeumen J, De Vries GE, Harder W, Dijkhuizen L (1991) Purification and characterization of an activator protein for methanol dehydrogenase from thermotolerant Bacillus spp., J Biol Chem 266:3955–3960
Bairoch A (1992) PROSITE: a dictionary of sites and patterns in proteins. Nucleic Acids Res 20:2013–2018
Bruchhaus I, Tannich E (1994) Purification and molecular characterization of the NAD+-dependent acetaldehyde/alcohol dehydrogenase from Entamoeba histolytica. Biochem J 303: 743–748
Bystrykh LV, Govorukhina NI, Van Ophem PW, Hektor HJ, Dijkhuizen L, Duine JA (1993a) Formaldehyde dismutase activities in gram-positive bacteria oxidizing methanol. J Gen Microbiol 139:1979–1985
Bystrykh LV, Vonck J, Van Bruggen EFJ, Van Beeumen J, Samyn B, Govorukhina NI, Arfman N, Duine JA, Dijkhuizen L (1993b) Electron microscopic analysis and structural characterization of novel NADP(H)-containing methanol: N,N′-dimethyl-4-nitrosoaniline oxidoreductases from the gram-positive methylotrophic bacteria Amycolatopsis methanolica and Mycobacterium gastri MB19. J Bacteriol 175:1814–1822
Carrey EA (1990) Peptide mapping. In: Creighton TE (ed) Protein structure: a practical approach. IRL Press, Oxford, pp 117–167
Conway T, Ingram LO (1989) Similarity of Escherichia coli propanediol oxidoreductase (fucO product) and an unusual alcohol dehydrogenase from Zymomonas mobilis and Saccharomyces cerevisiae. J Bacteriol 171:3754–3759
Conway T, Sewell GW, Osman YA, Ingram LO (1987) Cloning and sequencing of the alcohol dehydrogenase II gene from Zymomonas mobilis. J Bacteriol 169:2591–2597
Daniel R, Boenigk R, Gottschalk G (1995) Purification of 1,3-propanediol dehydrogenase from Citrobacter freundii and cloning, sequencing, and overexpression of the corresponding gene in Escherichia coli. J Bacteriol 177:2151–2156
De Mot R, Vanderleyden J (1989) Application of two-dimensional protein analysis for strain fingerprinting and mutant analysis of Azospirillum species. Can J Microbiol 35:960–967
De Mot R, Nagy I, Schoofs G, Vanderleyden J (1994) Sequence of a Rhodococcus gene cluster encoding the subunits of ethanolamine ammonia-lyase and an APC-like permease. Can J Microbiol 40:403–407
De Vries GE, Kües U, Stahl U (1990) Physiology and genetics of methylotrophic bacteria. FEMS Microbiol Rev 75:57–102
De Vries GE, Arfman N, Terpstra P, Dijkhuizen L (1992) Cloning, expression, and sequence analysis of the Bacillus methanolicus C1 methanol dehydrogenase gene. J Bacteriol 174:5346–5353
Dunn MF, Bernhard SA (1971) Rapid kinetic evidence for adduct formation between the substrate analog p-nitroso-N,N-dimethylaniline and reduced nicotinamide-adenine dinucleotide during enzymic reduction. Biochemistry 10:4569–4575
Elvin CM, Thompson PR, Argall ME, Hendry P, Stamford NPJ, Lilley PE, Dixon NE (1990) Modified bacteriophage lambda promoter vectors for overproduction of proteins in Escherichia coli. Gene 87:123–126
Fischer RJ, Helms J, Dürre P (1993) Cloning, sequencing, and molecular analysis of the sol operon of Clostridium acetobutylicum, a chromosomal locus involved in solventogenesis. J Bacteriol 175:6959–6969
Goodfellow M, Thomas EG, Ward AC, James AL (1990) Classification and identification of Rhodococci. Zentrabl Bakteriol 274:299–315
Henikoff S, Henikoff JG (1994) Protein family classification based on searching a database of blocks. Genomics 19:97–107
Jörnvall H, Persson B, Jeffery J (1987) Characteristics of alcohol/polyol dehydrogenase. The zinc-containing long-chain alcohol dehydrogenases. Eur J Biochem 167:195–201
Kessler D, Leibrecht I, Knappe J (1991) Pyruvate-formate-lyase-deactivase and acetyl-CoA reductase activities of Escherichia coli reside on a polymeric protein particle encoded by adhE. FEBS Lett 281:59–63
LeGendre N, Matsudaira PT (1989) Purification of proteins and peptides by SDS-PAGE. In: Matsudaira PT (ed) A practical guide to protein and peptide purification for microsequencing. Academic Press, San Diego, pp 49–69
Nagy I, Nagy J, Matyas J, Kecskés M (1987) Decomposition of EPTC by soil microbes in two soils. In: Proceedings of the British Crop Protection Conference (Weeds, vol 1). Lavenham Press, Lavenham, pp 525–530
Nagy I, Compernolle F, Ghys K, Vanderleyden J, De Mot R (1995a) A single cytochrome P-450 system is involved in degradation of the herbicides EPTC (S-ethyl dipropylthiocarbamate) and atrazine by Rhodococcus sp. NI86/21. Appl Environ Microbiol (in press)
Nagy I, Schoofs G, Compernolle F, Proost P, Vanderleyden J, De Mot R (1995b) Degradation of the thiocarbamate herbicide EPTC (S-ethyl dipropylcarbamothioate) and biosafening by Rhodococcus sp. strain NI86/21 involve an inducible cytochrome P-450 system and aldehyde dehydrogenase. J Bacteriol 177:676–687
Pearson WR (1990) Rapid and sensitive sequence comparison with FASTP and FASTA. Methods Enzymol 183:63–98
Reid MF, Fewson CA (1994) Molecular characterization of microbial alcohol dehydrogenases. Crit Rev Microbiol 20:13–56
Shields DC, Higgins DG, Sharp PM (1992) GCWIND: a microcomputer program for identifying open reading frames according to codon positional G+C content. Comp Appl Biosci 8:521–523
Sofia HJ, Burland V, Daniels DL, Plunkett G III, Blattner FR (1994) Analysis of the Escherichia coli genome V. DNA sequence of the region from 76.0 to 81.5 minutes. Nucleic Acids Res 22:2576–2586
Soubrier F, Lévy-Schil S, Mayaux J-F, Pétré D, Arnaud A, Crouzet J (1992) Cloning and primary structure of the widespectrum amidase from Brevibacterium sp. R312: high homology to the amiE product from Pseudomonas aeruginosa. Gene 116:99–104
Van Ophem PW, Van Beeumen J, Duine JA (1993) Nicotinoprotein [NAD(P)-containing] alcohol/aldehyde oxidoreductases. Purification and characterization of a novel type from Amycolatopsis methanolica. Eur J Biochem 212:819–826
Vonck J, Arfman N, De Vries GE, Van Beeumen J, Van Bruggen EFJ, Dijkhuizen L (1991) Electron microscopic analysis and biochemical characterization of a novel methanol dehydrogenase from the thermotolerant Bacillus sp. C1. J Biol Chem 266:3949–3954
Walter KA, Bennett GN, Papoutsakis ET (1992) Molecular characterization of two Clostridium acetobutylicum ATCC 824 butanol dehydrogenase isozyme genes. J Bacteriol 174:7149–7158
Williamson VM, Paquin CE (1987) Homology of Saccharomyces cerevisiae ADH4 to an iron-activated alcohol dehydrogenase from Zymomonas mobilis. Mol Gen Genet 209:374–381
Yang W, Li E, Kairong T, Stanley SL Jr (1994) Entamoeba histolytica has an alcohol dehydrogenase homologous to the multifunctional adhE gene product of Escherichia coli. Mol Biochem Parasitol 64:253–260
Youngleson JS, Jones WA, Jones DT, Woods DR (1989) Molecular analysis and nucleotide sequence of the adh1 gene encoding an NADPH-dependent butanol dehydrogenase in the Grampositive anaerobe Clostridium acetobutylicum. Gene 78:355–364
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Nagy, I., Verheijen, S., De Schrijver, A. et al. Characterization of the Rhodococcus sp. NI86/21 gene encoding alcohol: N,N′-dimethyl-4-nitrosoaniline oxidoreductase inducible by atrazine and thiocarbamate herbicides. Arch. Microbiol. 163, 439–446 (1995). https://doi.org/10.1007/BF00272133
Received:
Accepted:
Issue Date:
DOI: https://doi.org/10.1007/BF00272133