1887

Abstract

The -leucine dehydrogenase (LeuDH, EC 1.4.1.9) from the extremely thermophilic (DSM 405) has been purified to homogeneity and crystallized. Its physical and biochemical properties, such as subunit structure, , amino acid composition, isoelectric point, heat stability, pH-optima, values for coenzymes and substrates, and pattern of inhibition have been determined. The native enzyme is an octamer ( 320000) composed of identical subunits ( 39000) which contain one intrachain disulphide bridge. Only aliphatic amino acids were accepted as substrates and a preference was exhibited for branched-chain residues. Inhibition occurred only upon incubation with thiol reagents such as HgCl and -mercuribenzoate, and pyridoxal 5'-phosphate. The LeuDH was very thermostable, with 50% residual activity remaining after 30 min incubation at 80 C. Its properties, as well as amino acid composition and N-terminal sequence, were compared with those of the phylogenetically related LeuDH from the mesophile . Both enzymes displayed very similar properties: the quaternary structures were identical, amino acid composition alike, the N-terminal sequence showed 62% homology for the first 19 residues, and values for the different substrates differed by a factor of two or less, indicating that the active centres had a similar structure. Thus, the only major difference observed was the much higher stability of the thermophilic enzyme to denaturation by heat and organic solvents.

Loading

Article metrics loading...

/content/journal/micro/10.1099/00221287-135-5-1305
1989-05-01
2022-01-17
Loading full text...

Full text loading...

/deliver/fulltext/micro/135/5/mic-135-5-1305.html?itemId=/content/journal/micro/10.1099/00221287-135-5-1305&mimeType=html&fmt=ahah

References

  1. Argos P., Rossmann M.G., Grau U.M., Zuber H., Frank G., Tratschin J.D. 1979; Thermal stability and protein structure. Biochemistry 18:5698–5703
    [Google Scholar]
  2. Asano Y., Nakazawa A., Endo K., Hibino Y., Ohmori M., Numao N., Kondo K. 1987; Phenylalanine dehydrogenase ofBacillus badius . European Journal of Biochemistry 168:153–159
    [Google Scholar]
  3. Barstow D.A., Murphy J.P., Sharman A.F., Clarke A.R., Holbrook J.J., Atkinson T. 1987; Amino acid sequence of the l-lactate dehydrogenase ofBacillus caldotenax deduced from the nucleotide sequence of the cloned gene. European Journal of Biochemistry 165:581–586
    [Google Scholar]
  4. Butcher L.A., Tomkins J.K. 1986; A comparison of silver staining methods for detecting proteins in ultrathin polyacrylamide gels on support film after isoelectric focusing. Analytical Biochemistry 148:384–388
    [Google Scholar]
  5. Friedrich B., Heine E., Finck A., Friedrich C.G. 1981; Nickel requirement for active hydro- genase formation inAlcaligenes eutrophus . Journal of Bacteriology 145:1144–1149
    [Google Scholar]
  6. Fujita T., Suzuki Y., Yamauti S-I., Takagahara I., Fujii K., Yamashita J., Horio T. 1980; Chromatography in the presence of high concentrations of salts on columns of celluloses with and without ion exchange groups (hydrogen bond chromatography). Journal of Biochemistry 87:89–100
    [Google Scholar]
  7. Harris C.E., Teller D.C. 1973; Estimation of primary sequence homology from amino acid composition of evolutionary related proteins. Journal of Theoretical Biology 38:347–362
    [Google Scholar]
  8. Harris C.E., Kobes R.D., Teller D.C., Rutter W.J. 1969; The molecular characteristics of yeast aldolase. Biochemistry 8:2442–2454
    [Google Scholar]
  9. Hediger M.A., Frank G., Zuber H. 1986; The primary structure of the mesophilic lactate dehydrogenase fromBacillus subtilis . Biological Chemistry Hoppe-Seyler 367:891–903
    [Google Scholar]
  10. Hirs C.H.W. 1967; Performic acid oxidation. Methods in Enzymology 11:197–199
    [Google Scholar]
  11. Kalb V.F. Jr Bernlohr R.W. 1977; A new spectrophotometric assay for protein in cell extracts. Analytical Biochemistry 82:362–371
    [Google Scholar]
  12. Kärst U., Tsai H., Schütte H. 1986a; Conserva-tion of antigenic determinants in leucine dehydrogenase from thermophilic and mesophilic Bacillus strains . FEMS Microbiology Letters 37:335–339
    [Google Scholar]
  13. Kärst U., Schütte H., Tsai H. 1986b; Detection in thermophilicBacillus strains of a leucine dehydrogenase immunologically related to the enzyme from Bacillus cereus . Biological Chemistry Hoppe-Seyler 367 Suppl.,335
    [Google Scholar]
  14. Kärst U., Schütte H., Baydoun H., Tsai H. 1987; Purification and properties of l-alanine and l-leucine dehydrogenases from the thermophilicBacillus caldolyticus . Proceedings, 4th European Congress on Biotechnology 2: pp 220–223 Amsterdam:: Elsevier.;
    [Google Scholar]
  15. Keradjopoulos D., Holldorf A.W. 1979; Purification and properties of alanine dehydrogenase fromHalobacterium salinarium . Biochimica et biophysica acta 570:1–10
    [Google Scholar]
  16. Lünsdorf H., Tsai H. 1985; Quaternary organisation of subunits in the l-leucine dehydrogenase fromBacillus cereus . FEBS Letters 193:261–266
    [Google Scholar]
  17. Misono H., Soda K. 1980; Properties ofmeso-α-σdiaminopimelate d-dehydrogenase from Bacillus sphaericus . Journal of Biological Chemistry 255:10599–10605
    [Google Scholar]
  18. Obermeier N., Poralla K. 1976; Some physiological functions of the l-leucine dehydrogenase inBacillus subtilis . Archives of Microbiology 109:59–63
    [Google Scholar]
  19. Ohshima T., Soda K. 1984; Modification of leucine dehydrogenase by pyridoxal 5′-phosphate. Agricultural and Biological Chemistry 48:349–354
    [Google Scholar]
  20. Ohshima T., Nagata S., Soda K. 1985a; Purification and characterisation of thermostable leucine dehydrogenase from Bacillus stearothermophilus . Archives of Microbiology 141:407–411
    [Google Scholar]
  21. Ohshima T., Misono H., Soda K. 1978a; Properties of crystalline leucine dehydrogenase from Bacillus sphaericus . Journal of Biological Chemistry 253:5719–5725
    [Google Scholar]
  22. Ohshima T., Yamamoto T., Misono H., Soda K. 1978b; Leucine dehydrogenase ofBacillus sphaericus: sulfhydryl groups and catalytic sites. Agricultural and Biological Chemistry 42:1739–1743
    [Google Scholar]
  23. Ohshima T., Wandrey C., Sugiura M., Soda K. 1985b; Screening of thermostable leucine and alanine dehydrogenases in thermophilicBacillusstrains. Biotechnology Letters 7:871–876
    [Google Scholar]
  24. Poralla K. 1971; The induction of a dehydrogenase activity for branched chain amino acids inBacillus subtilis . Archives of Microbiology 77:339–343
    [Google Scholar]
  25. Priest F.G. 1981; DNA homology in the genusBacillus . In The Aerobic Endospore-forming Bacteria : Classification and Identification pp 33–57 Berkeley R. C. W., Goodfellow M. Edited by London: Academic Press;
    [Google Scholar]
  26. Priest F.G., Goodfellow M., Todd C. 1981; The genusBacillus: A numerical analysis. In The Aerobic Endospore-forming Bacteria: Classification and Identification pp 91–103 Berkeley R. C. W., Goodfellow M. Edited by London: Academic Press;
    [Google Scholar]
  27. Riddles P.W., Blakeley R.L., Zerner B. 1983; Reassessment of Ellman’s reagent. Methods in Enzymology 91:49–60
    [Google Scholar]
  28. Rothe G.M. 1982; Applicability of the log MM - Drelationship to linear polyacrylamide gradient gel electrophoresis under a wide range of experimental conditions. Electrophoresis 3:255–262
    [Google Scholar]
  29. Rothe G.M., Purkhanbaba H. 1982; Determination of molecular weights and Stokes’ radii of nondenatured proteins by polyacrylamide gradient gel electrophoresis. 1. An equation relating total polymer concentration, the molecular weight of proteins in the range of 104-106, and duration of electrophoresis. Electrophoresis 3:33–42
    [Google Scholar]
  30. Schlatter D., Krieck O., Suter F., Zuber H. 1987; The primary structure of the psychrophilic lactate dehydrogenase fromBacillus psychrosacchar-olyticus . Biological Chemistry Hoppe-Seyler 368:1435–1446
    [Google Scholar]
  31. Schütte FL, Tsai H., Kula M.-R. 1985; l-Leucine dehydrogenase fromBacillus cereus. Production, large-scale purification and protein characterisation. Applied Microbiology and Biotechnology 22:306–317
    [Google Scholar]
  32. Smith E.L., Austen B.M., Blumenthal K.M., Nyc J.F. 1975; Glutamate dehydrogenases. In The Enzymes. 11: pp 293–367 Boyer P. D. Edited by New York: Academic Press;
    [Google Scholar]
  33. Smith T.F., Waterman M.S. 1981; Identification of common molecular subsequences. Journal of Molecular Biology 147:195–197
    [Google Scholar]
  34. Suzuki Y., Yasui T., Mino Y., Abe S. 1987; A strong correlation between the increase in number of proline residues and the rise in thermostability of fiveBacillus oligo-l,6-glucosidases. Applied Microbiology and Biotechnology 11:23–27
    [Google Scholar]
  35. Wandrey C., Bossow B. 1986; Continuous cofactor regeneration. Utilization of polymer bound NAD(H) for the production of optically active acids. Bioteknologiya & Biotekhnika 3:8–13
    [Google Scholar]
  36. Wandrey C., Wichmann R., Berke W., Morr M., Kula M.-R. 1984; Continuous cofactor regeneration in membrane reactors. Proceedings, 3rd European Congress on Biotechnology I: pp. 239–244 Weinheim:: Verlag Chemie.;
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/micro/10.1099/00221287-135-5-1305
Loading
/content/journal/micro/10.1099/00221287-135-5-1305
Loading

Data & Media loading...

Most cited this month Most Cited RSS feed

This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error