The Biochemical Characterization of D-Hydroxyisovalerate Dehydrogenase, a Key Enzyme in the Biosynthesis of Enniatins

  • Lee, Chan (Department of Food Science and Technology, Chung-ang University) ;
  • Zocher, Rainer (Institut fuer Biochemie und Molekulare Biologie, Technischen Universitaet Berlin)
  • 투고 : 1996.07.10
  • 발행 : 1996.11.30

초록

The biochemical properties of purified D-hydruxyisovalerate dehydrogenase from Fusarium sambucinum was elucidated. D-Hydroxyisovalerate dehydrogenase produced solely D-hydroxyisovalerate from 2-ketoisovalerate. The isoelectric point of the purified enzyme was 7.0. The enzyme was highly specific with 2-ketoisovalerate ($K_{m}=0.188$ mM, $V_{max}=8.814$ mmol/min mg) and 2-keto-3-methyl-n-valerate ($K_{m}=0.4$ mM, $V_{max}=1.851$ mmol/min mg) for the reductive reaction. This was also seen by comparing D-hydroxyisovalerate ($K_{m}=1.667$ mM, $V_{max}=0.407$ mmol/min mg) and D-hydroxy-3-methyl-n-valerate ($K_{m}=6.7$ mM, $V_{max}=0.648$ mmol/min mg) for the oxidative reaction. Thiol blocking reagents, such as iodoacetamide, N-ethylmaleimide and p-chloromecuribenzoate inhibited about 80% of enzyme activity at 0.02 mM, 50 mM and 50 mM, respectively. The enzyme activity was also inhibited by the addition of 0.1 mM of various metal ions, such as $Fe^{2+}$ (67%), $Cu^{2+}$ (88%), $Zn^{2+}$ t (76%) and $Mg^{2+}$ (9%). The enzyme was stable over three months in 50 mM potassium phosphate buffer (pH 5~7) at $-80^{\circ}C$. However the purified enzyme lost 30% of its activity in the same buffer after 24 h at $4^{\circ}C$. The studies about thermal inactivation of D-hydroxyisovalerate dehydrogenase exhibit 209.2 kJ/M of activation enthalpy and 0.35 kJ/mol K of activation entropy.

키워드

참고문헌

  1. Can. J. Microbial. v.19 Audhya, T.K.;Russel, D.W. https://doi.org/10.1139/m73-166
  2. Biochemistry v.26 Billich, A.;Zocher, R. https://doi.org/10.1021/bi00399a058
  3. Electrophoresis v.8 Blum, H.;Beiger, H.;Gross, H.J. https://doi.org/10.1002/elps.1150080203
  4. Anal. Biochem. v.72 Bradford, M.M. https://doi.org/10.1016/0003-2697(76)90527-3
  5. Dryfuss, M.M.;Schreier, M.H.;Tscherter;Wenger, R.
  6. J. Chem. Phys. v.3 Eyring, H. https://doi.org/10.1063/1.1749604
  7. J. Chromatogr. v.448 Guenter. K. https://doi.org/10.1016/S0021-9673(01)84562-3
  8. Eur. J. Appl. Microbiol. Biotechnol. v.18 Hummel, W.;Schuette, H.;Kula, M.R. https://doi.org/10.1007/BF00500828
  9. Appl. Microbiol. Biotechnol. v.21 Hummel, W.;Schuette, H.;Kula, M.R.
  10. Nature v.227 Laemmmli, U.K. https://doi.org/10.1038/227680a0
  11. J. Biol. Chem. v.265 Lawen, A.;Zocher, R.
  12. J. Biol. Chem. v.267 Lee, C.;Goerisch, H.;Kleinkauf, H.;Zocher, R.
  13. Eur. J. Microbiol Biotechnol. v.17 Madry, N.;Zocher, R.;Kleinkauf. H. https://doi.org/10.1007/BF00499854
  14. J. Biol. Chem. v.184 Meister, A.
  15. Peptidlactones, in Biochemistry and Genetic Regulation of Commercially Important Antibiotics Okumura, Y.;Vining, L.C.(ed.)
  16. Phytochemistry v.20 Pais, M.;Das, B.C.;Ferron, P. https://doi.org/10.1016/0031-9422(81)85160-6
  17. J. Antibiot. v.36 Peeters, H.;Zocher, R.;Madry, N.;Oelrichs, P.B.;Kleinkauf, H.;Kraepelin, G. https://doi.org/10.7164/antibiotics.36.1762
  18. J. Antibiot. v.45 Pieper, R.;Kleinkauf, H.;Zocher, R. https://doi.org/10.7164/antibiotics.45.1273
  19. Appl. Microbiol. Biotechnol. v.19 Schuette, H.;Hummel, W.;Kula, M.R. https://doi.org/10.1007/BF00256449
  20. J. Am. Chem. Soc. v.97 Smith, G.D.;Duax, W.L.;Langs, D.A.;Detitta, G.T.;Edmonds, J.W.;Rohrer, D.C.;Weeks, C.M. https://doi.org/10.1021/ja00858a008
  21. Adv. Enzymol. v.9 Stern, A.E.
  22. FEBS Lett. v.71 Zocher, R.;Salinikow, J.;Kleinkauf, H. https://doi.org/10.1016/0014-5793(76)80887-3
  23. Biochemistry v.21 Zocher, R.;Keller, U.;Kleinkauf, H. https://doi.org/10.1021/bi00530a008
  24. Biochem. Biophys. Res. Commun. v.110 Zocher, R.;Keller, U.;Kleinkauf, H. https://doi.org/10.1016/0006-291X(83)91294-9