Abstract
An NAD+-dependent alcohol dehydrogenase of a psychrotorelant from Antarctic seawater, Flavobacterium frigidimaris KUC-1 was purified to homogeneity with an overall yield of about 20% and characterized enzymologically. The enzyme has an apparent molecular weight of 160k and consists of four identical subunits with a molecular weight of 40k. The pI value of the enzyme and its optimum pH for the oxidation reaction were determined to be 6.7 and 7.0, respectively. The enzyme contains 2 gram-atoms Zn per subunit. The enzyme exclusively requires NAD+ as a coenzyme and shows the pro-R stereospecificity for hydrogen transfer at the C4 position of the nicotinamide moiety of NAD+. F. frigidimaris KUC-1 alcohol dehydrogenase shows as high thermal stability as the enzymes from thermophilic microorganisms. The enzyme is active at 0 to over 85°C and the most active at 70°C. The half-life time and k cat value at 60°C were calculated to be 50 min and 27,400 min−1, respectively. The enzyme also shows high catalytic efficiency at low temperatures (0–20°C) (k cat/K m at 10°C; 12,600 mM−1 min−1) similar to other cold-active enzymes from psychrophiles. The alcohol dehydrogenase gene is composed of 1,035 bp and codes 344 amino acid residues with an estimated molecular weight of 36,823. The sequence identities were found with the amino acid sequences of alcohol dehydrogenases from Moraxella sp. TAE123 (67%), Pseudomonas aeruginosa (65%) and Geobacillus stearothermophilus LLD-R (56%). This is the first example of a cold-active and thermostable alcohol dehydrogenase.
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Abbreviations
- AlcDH:
-
Alcohol dehydrogenase
- CHES:
-
N-cyclohexyl-2-aminoethanesulfonic acid
- conc.:
-
Concentration
- KPB:
-
Potassium phosphate buffer
- LMW:
-
Low molecular weight
- PVDF:
-
Poly (vinylidene fluoride)
- Tris:
-
Tris (hydroxymethyl) aminomethane
- UPGMA:
-
Unweighted pair-group method with arithmetic mean
References
Akeson A (1964) On the zinc content of horse liver alcohol dehydrogenase. Biochem Biophys Res Commun 17:211–214
Bergeron H, Labbe D, Turmel C, Lau PC (1998) Cloning, sequence and expression of a linear plasmid-based and a chromosomal homolog of chloroacetaldehyde dehydrogenase-encoding genes in Xanthobacter autotrophicus GJ10. Gene 19:9–18
Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254
Cannio R, Rossi M, Bartolucci S (1994) A few amino acid substitutions are responsible for the higher thermostability of a novel NAD+-dependent bacillar alcohol dehydrogenase. Eur J Biochem 222:345–352
Cheng LY, Lek LH (1992) Inhibition of alcohol dehydrogenases by thiol compounds. FEBS Lett 300:251–253
Coker JA, Sheridan PP, Curtze JL, Gutshall KR, Auman AJ, Brenchley JE (2003) Biochemical characterization of a β-galactosidase with a low temperature optimum obtained from an Antarctic Arthrobacter isolate. J Bacteriol 185:5473–5482
Drum DE, Harrison JH 4th, Li TK, Bethune JL,Vallee BL (1967) Structural and functional zinc in horse liver alcohol dehydrogenase. Proc Natl Acad Sci USA 57:1434–1440
Eklund H, Plapp BV, Samama JP, Branden CI (1982) Binding of substrate in a ternary complex of horse liver alcohol dehydrogenase. J Biol Chem 257:14349–14358
Esaki N, Shimoi H, Nakajima N, Ohshima T, Tanaka H, Soda K (1989) Enzymatic in situ determination of stereospecificity of NAD-dependent dehydrogenases. J Biol Chem 264:9750–9752
Fan F, Lorenzen JA, Plapp BV (1991) An aspartate residue in yeast alcohol dehydrogenase I determines the specificity for coenzyme. Biochemistry 30:6397–6401
Feller G, Gerday C (1997) Psychrophilic enzymes: molecular basis of cold adaptation. Cell Mol Life Sci 53:830–841
Feller G, Gerday C (2003) Psychrophilic enzymes: hot topics in cold adaptation. Nature Rev 130–132:200–208
Fields PA, Somero GN (1998) Hot spots in cold adaptation: localized increases in conformational flexibility in lactate dehydrogenase A4 orthologs of Antarctic notothenioid fishes. Proc Natl Acad Sci USA 95:11476–11481
Fujiwara S (2002) Extremophiles: developments of their special functions and potential resources. J Biosci Bioeng 94:518–525
Guagliardi A, Martino M, Iaccarino I, Rosa MDe, Rossi M, Bartolucci S (1996) Purification and characterization of the alcohol dehydrogenase from a novel strain of Bacillus stearothermophilus growing at 70 degrees C. Int J Biochem Cell Biol 28:239–246
Hirakawa H, Kamiya N, Kawarabayashi Y, Nagamune T (2004) Properties of an alcohol dehydrogenase from the hyperthermophilic archaeon Aeropyrum pernix K1. J Biosci Bioeng 97:202–206
Innis MA, Myambo KB, Gelfand DH, Brow MAD (1988) DNA sequencing with Thermus aquaticus DNA polymerase and direct sequencing of polymerase chain reaction-amplified DNA. Proc Natl Acad Sci USA 85:9436–9440
Jornvall H, Hoog JO, Lindstrom HB, Vallee BL (1987) Mammalian alcohol dehydrogenases of separate classes: intermediates between different enzymes and intraclass isozymes. Proc Natl Acad Sci USA 84:2580–2584
Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685
LeBrun LA, Park DH, Ramaswamy S, Plapp BV (2004) Participation of histidine-51 in catalysis by horse liver alcohol dehydrogenase. Biochemistry 43:3014–3026
Leskovac V, Trivic S, Pericin D (2002) The three zinc-containing alcohol dehydrogenases from baker’s yeast, Saccharomyces cerevisiae. FEMS Yeast Res 2:481–494
Li D, Stevenson KJ (1997) Purification and sequence analysis of a novel NADP(H)-dependent type III alcohol dehydrogenase from Thermococcus strain AN1. J Bacteriol 179:4433–4437
Liang ZX, Lee T, Resing KA, Ahn NG, Klinman JP (2004a) Thermal-activated protein mobility and its correlation with catalysis in thermophilic alcohol dehydrogenase. Proc Natl Acad Sci USA 101:9556–9561
Liang ZX, Tsigos I, Lee T, Bouriotis V, Resing KA, Ahn NG, Klinman JP (2004b) Evidence for increased local flexibility in psychrophilic alcohol dehydrogenase relative to its thermophilic homologue. Biochemistry 43:14676–14683
Lonhienne T, Gerday C, Feller G (2000) Psychrophilic enzymes: revising the thermodynamic parameters of activation may explain local flexibility. Biochim Biophys Acta 1543:1–10
Nakajima N, Nakamura K, Esaki N, Tanaka H, Soda K (1989) Enzymatic in situ analysis by 1H-NMR of the hydrogen transfer stereospecificity of NAD(P)+-dependent dehydrogenases. J Biochem 106:515–517
Nogi Y, Soda K, Oikawa T (2005) Flavobacterium frigidimaris sp. nov., isolated from Antarctic seawater. Syst Appl Microbiol 28:310–315
Ohshima T, Soda K (1979) Purification and properties of alanine dehydrogenase from Bacillus sphaericus. Eur J Biochem 100:29–30
Ohshima T, Sakane M, Yamazaki T, Soda K (1990) Thermostable alanine dehydrogenase from thermophilic Bacillus sphaericus DSM462: purification, characterization and kinetic mechanism. Eur J Biochem 191:715–720
Oikawa T, Yamanaka K, Kazuoka T, Kanzawa N, Soda K (2001) Psychrophilic valine dehydrogenase of the antarctic psychrophile, Cytophaga sp. KUC-1: purification, molecular characterization and expression. Eur J Biochem 268:4375–4383
Peretz M, Bogin O, Keinan E, Burstein Y (1993) Stereospecificity of hydrogen transfer by the NADP-linked alcohol dehydrogenase from the thermophilic bacterium Thermoanaerobium brockii. Int J Pept Protein Res 42:490–495
Priefert H, Kruger N, Jendrossek D, Schmidt B, Steinbuchel A (1992) Identification and molecular characterization of the gene coding for acetaldehyde dehydrogenase II (acoD) of Alcaligenes eutrophus. J Bacteriol 174:899–907
Radianingtyas H, Wright PC (2003) Alcohol dehydrogenase from thermophilic and hyperthermophilic archaea and bacteria. FEMS Microb Rev 27:593–616
Russell NJ (1998) Molecular adaptations in psychrophilic bacteria: potential for biotechnological applications. In: Scheper T (ed) Advances in biochemical engineering biotechnology, vol. 61, Springer, Berlin Heidelberg New York, pp 1–21
Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual. 2nd edn, Cold Spring Harbor Laboratory, Cold Spring Harbor
Sekhar VC, Plapp BV (1988) Mechanism of binding of horse liver alcohol dehydrogenase and nicotinamide adenine dinucleotide. Biochemistry 27:5082–5088
Svingor A, Kardos J, Hajdu I, Nemeth A, Zavodszky P (2001) A better enzyme to cope with cold. Comparative flexibility studies on psychrotrophic, mesophilic, and thermophilic IPMDHs. J Biol Chem 276:28121–28125
Theorell H, Chance B (1951) Liver alcohol dehydrogenase. II. Kinetics of the compound of horse-liver alcohol dehydrogenase and reduced diphospho pyridene nucleotide. Acta Chem Scand 5:1127–1144
Tsigos I, Velonia K, Smonou I, Bouriotis V (1998) Purification and characterization of an alcohol dehydrogenase from the Antarctic psychrophile Moraxella sp. TAE123. Eur J Biochem 254:356–362
Tulchin N, Ornstein L, Davis BJ (1976) A microgel system for disc electrophoresis. Anal Biochem 72:485–490
Velonia K, Tsigos I, Bouriotis V, Smonou I (1999) Stereospecificity of hydrogen transfer by the NAD(+)-linked alcohol dehydrogenase from the Antarctic psychrophile Moraxella sp. TAE123. Bioorg Med Chem Lett 9:65–68
Vieille C, Zeikus GJ (2001) Hyperthermophilic enzymes: sources, uses, and molecular mechanisms for thermostability. Microbiol Mol Biol Rev 65:1–43
Weinhold EG, Glasfeld A, Ellington AD, Benner SA (1991) Structural determinants of stereospecificity in yeast alcohol dehydrogenase. Proc Natl Acad Sci USA 88:8420–8424
Wiedmann M, Weilmeier D, Dineen SS, Ralyea R, Boor KJ (2000) Molecular and phenotypic characterization of Pseudomonas spp. isolated from milk. Appl Environ Microbiol 66:2085–2095
Yamanaka Y, Kazuoka T, Yoshida M, Yamanaka K, Oikawa T, Soda K (2002) Thermostable aldehyde dehydrogenase from psychrophile, Cytophaga sp. KUC-1: enzymological characteristics and functional properties. Biochem Biophys Res Commun 298:632–637
Yumoto I, Iwata H, Sawabe T, Ueno K, Ichise N, Matsuyama H, Okuyama H, Kawasaki K (1999) Characterization of a facultatively psychrophilic bacterium, Vibrio rumoiensis sp. nov., that exhitits high catalase activity. Appl Environ Microbiol 65:67–72
Acknowledgments
This research was partially supported by the Ministry of Education, Culture, Sports, Science, and Technology (MEXT) of Japan, Grant-in-Aid for Scientific Research (C), 2004, No.16550150, and the High-Tech Research Center project for Private Universities, matching fund subsidy from MEXT (2002–2006), and the Kansai University Special Research Fund (2005).
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Kazuoka, T., Oikawa, T., Muraoka, I. et al. A cold-active and thermostable alcohol dehydrogenase of a psychrotorelant from Antarctic seawater, Flavobacterium frigidimaris KUC-1. Extremophiles 11, 257–267 (2007). https://doi.org/10.1007/s00792-006-0034-1
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DOI: https://doi.org/10.1007/s00792-006-0034-1