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Molecular analysis of an NAD-dependent alcohol dehydrogenase from the zygomycete Mucor circinelloides

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NAD-dependent alcohol dehydrogenase (ADH) activity was detected mainly in the cytosol of aerobically cultured mycelium and in anaerobically grown yeast cells of Mucor circinelloides. ADH levels were about 2.5-fold higher in yeast cells than in mycelium; zymogram analysis suggested that the same ADH enzyme is produced in both developmental stages. The enzyme, named ADH1, was purified to homogeneity from yeast cells, using ion- exchange and affinity chromatography. The active ADH1 appears to be a homomeric tetramer of 37,500-kDa subunits. K m values obtained for acetaldehyde, ethanol, NADH and NAD+ indicated that in vivo the enzyme mainly serves to reduce acetaldehyde to ethanol. Amino acid sequences of internal peptides obtained from the purified ADH1 were used to design oligonucleotides that allowed the cloning of the corresponding cDNA by RT-PCR, and the characterization of the genomic DNA sequence. The adh1 ORF is interrupted by two small introns located towards the 5′-end. M. circinelloides adh1 encodes a protein of 348 amino acids, which display moderate to high overall identity to several hypothetical ADH enzymes from the related zygomycete Rhizopus oryzae. adh1 mRNA is expressed at similar levels in aerobic mycelium and anaerobic yeast cells. During exponential growth under aerobic conditions, the level of adh1 transcript was correlated with the glucose concentration in the growth medium.

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  1. 1.

    Aljabani SM, Martinez I (1997) Universal and rapid salt-extraction of high quality genomic DNA for PCR-based techniques. Nucleic Acids Res 25:4692–4693

  2. 2.

    Ballance DJ (1986) Sequences important for gene expression in filamentous fungi. Yeast 2:229–236

  3. 3.

    Bartnicki-Garcia S (1963) Symposium on biochemical bases of morphogenesis in fungi III Mold-yeast dimorphism of Mucor. Bacteriol Rev 27:293–304

  4. 4.

    Bartnicki-Garcia S, Nickerson WJ (1962) Nutrition, growth and morphogenesis of Mucor rouxii. J Bacteriol 84:841–858

  5. 5.

    Borgia PT, Gokul NK, Phillips GJ (1985) Respiratory-competent conditional developmental mutant of Mucor racemosus. J Bacteriol 164:1049–1056

  6. 6.

    Boxma B, Voncken F, Jannink S, van Alen T, Akhmanova A, van Weelden SW, van Hellemond JJ, Ricard G, Huynen M, Tielens AG, Hackstein JH (2004) The anaerobic chytridiomycete fungus Piromyces sp E2 produces ethanol via pyruvate:formate lyase and an alcohol dehydrogenase E. Mol Microbiol 51:1389–1399

  7. 7.

    Ceccarelli C, Liang ZX, Strickler M, Prehna G, Goldstein BM, Klinman JP, Bahnson BJ (2004) Crystal structure and amide H/D exchange of binary complexes of alcohol dehydrogenase from Bacillus stearothermophilus: insight into thermostability and cofactor binding. Biochemistry 43:5266–5277

  8. 8.

    Dixon M, Webb EC (1979) Enzymes. Longman, London

  9. 9.

    Espinosa A, Clark D, Stanley SL (2004) Entamoeba histolytica alcohol dehydrogenase 2 (EhADH2) as a target for anti-amoebic agents. J Antimicrob Chemother 54:56–59

  10. 10.

    Felipe MSS, et al (2003) Transcriptome characterization of the dimorphic and pathogenic fungus Paracoccidioides brasiliensis by EST analysis. Yeast 20:263–271

  11. 11.

    Fernandez J, Gharahdaghi F, Mische SM (1998) Routine identification of proteins from SDS-PAGE gels or PVDF membranes using Matrix-Assisted-Laser-Desorption-Ionization Time-of Flight Mass Spectrometry. Electrophoresis 19:1036–1045

  12. 12.

    Jörnvall H, Persson B, Jeffery J (1987) Characteristics of alcohol/polyol dehydrogenases. Eur J Biochem 167:195–201

  13. 13.

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

  14. 14.

    Larroy C, Fernandez RM, Gonzalez E, Pares X, Biosca JA (2003) Properties and functional significance of Saccharomyces cerevisiae ADHVI. Chem Biol Interact 143–144:229–238

  15. 15.

    Lowry OH, Rosebrough NJ, Farr AL, Randall RJ (1951) Protein measurement with the Folin phenol reagent. J Biol Chem 193:265–275

  16. 16.

    Lübbehüsen TL, Nielsen J, McIntyre M (2004) Aerobic and anaerobic ethanol production by Mucor circinelloides during submerged growth. Appl Microbiol Biotechnol 63:543–548

  17. 17.

    McIntyre M, Breum J, Arnau J, Nielsen J (2002) Growth physiology and dimorphism of Mucor circinelloides (syn.racemosus) during submerged batch cultivation. Appl Microbiol Biotechnol 58:495–502

  18. 18.

    Nikolova P, Ward OP (1991) Production of L-phenylacetyl carbionol by biotransformation: product and by-product formation and activities of the key enzymes in wild-type and ADH isoenzyme mutants of Saccharomyces cerevisiae. Biotechnol Bioeng 20:493–498

  19. 19.

    Orlowski M (1991) Mucor dimorphism. Microbiol Rev 55:234–258

  20. 20.

    Palecek SP, Parikh AS, Kron SJ (2002) Sensing, signalling and integrating physical processes during Saccharomyces cerevisiae invasive and filamentous growth. Microbiol 148:893–907

  21. 21.

    Passoth V, Schafer B, Liebel B, Weierstall T, Klinner U (1998) Molecular cloning of alcohol dehydrogenase genes of the yeast Pichia stipitis and identification of the fermentative ADH. Yeast 14:1311–1325

  22. 22.

    Reid MF, Fewson CA (1994) Molecular characterization of microbial alcohol dehydrogenases. Crit Rev Microbiol 20:13–56

  23. 23.

    Roncero MIG (1984) Enrichment method for the isolation of auxotrophic mutants of Mucor using the polyene antibiotic N-glycosyl-polifungin. Carlsberg Res Commun 49:685–690

  24. 24.

    Salcedo HR, Ruiz-Herrera J (1993) Isolation and characterization of a mycelial cytochrome aa3-deficient mutant and the role of mitochondria in dimorphism of Mucor rouxii. Exp Mycol 17:142–154

  25. 25.

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

  26. 26.

    Sypherd P, Borgia PT, Paznokas JL (1978) Biochemistry of dimorphism in the fungus Mucor. Adv Microb Physiol 18:67–104

  27. 27.

    Torres-Guzman JC, Arreola-Garcia GA, Zazueta-Sandoval R, Carrillo-Rayas T, Martinez-Cadena G, Gutierrez-Corona F (1994) Genetic evidence for independence between fermentative metabolism (ethanol accumulation) and yeast-cell development in the dimorphic fungus Mucor rouxii. Curr Genet 26:166–171

  28. 28.

    Wierenga RK, Hol WG (1983) Predicted nucleotide binding properties of p21 protein and its cancer-associated variant. Nature 302:842–844

  29. 29.

    Wolff AM, Arnau J (2002) Cloning of glyceraldehyde-3-phosphate dehydrogenase-encoding genes in Mucor circinelloides (syn racemosus) and use of the gpd1 promoter for recombinant protein production. Fungal Genet Biol 35:21–29

  30. 30.

    Zazueta-Sandoval R, Gutierrez-Corona JF (1999) Developmental and environmental influences in the production of a single NAD-dependent fermentative alcohol dehydrogenase by the zygomycete Mucor rouxii. Arch Microbiol 172:280–286

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This work was supported by the Consejo Nacional de Ciencia y Tecnología (CONACyT), México and the University of Guanajuato. RARP and VMC were recipients of a fellowship from CONACyT, Mexico. The work has been carried out in compliance with the current laws governing genetic experimentation in Mexico

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Correspondence to J. F. Gutiérrez-Corona.

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Communicated by P. Punt

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Rangel-Porras, R.A., Meza-Carmen, V., Martinez-Cadena, G. et al. Molecular analysis of an NAD-dependent alcohol dehydrogenase from the zygomycete Mucor circinelloides . Mol Genet Genomics 274, 354–363 (2005). https://doi.org/10.1007/s00438-005-0025-4

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  • NAD-dependent alcohol dehydrogenase (ADH)
  • Reverse genetics
  • Mucor circinelloides
  • Dimorphism
  • Glucose metabolism
  • Gene cloning