Isolation and characterization of valine dehydrogenase from Streptomyces aureofaciens

Valine dehydrogenase was purified to homogeneity from the crude extracts of Streptomyces aureofaciens. The molecular weight of the native enzyme was 116,000 by equilibrium ultracentrifugation and 118,000 by size exclusion high-performance liquid chromatography. The enzyme was composed of four subunits with molecular weights of 29,000. The isoelectric point was 5.1. The enzyme required NAD+ as a cofactor, which could not be replaced by NADP+. Sulfhydryl reagents inhibited the enzyme activity. The pH optimum was 10.7 for oxidative deamination of L-valine and 9.0 for reductive amination of alpha-ketoisovalerate. The Michaelis constants were 2.5 mM for L-valine and 0.10 mM for NAD+. For reductive amination the Km values were 1.25 mM for alpha-ketoisovalerate, 0.023 mM for NADH, and 18.2 mM for NH4Cl.

[1]  J. Neuzil,et al.  Isolation of pure anhydrotetracycline oxygenase from Streptomyces aureofaciens. , 1988, The Biochemical journal.

[2]  T. Řezanka,et al.  Effect of ammonium ions on the composition of fatty acids in Streptomyces fradiae, producer of tylosin , 1987 .

[3]  U. Kärst,et al.  Conservation of antigenic determinants in leucine dehydrogenase from thermophilic and mesophilic Bacillus strains , 1986 .

[4]  S. Ōmura,et al.  Ammonium ion suppresses the biosynthesis of tylosin aglycone by interference with valine catabolism in Streptomyces fradiae. , 1983, The Journal of antibiotics.

[5]  P. E. Granum,et al.  An absolute method for protein determination based on difference in absorbance at 235 and 280 nm. , 1980, Analytical biochemistry.

[6]  K. Poralla,et al.  Experiments on the role of leucine dehydrogenase in initiation of Bacillus subtilis spore germination , 1979 .

[7]  T. Ohshima,et al.  Properties of crystalline leucine dehydrogenase from Bacillus sphaericus. , 1978, The Journal of biological chemistry.

[8]  Y. Nitta,et al.  l-Amino Acid Dehydrogenases in Bacillus subtilis Spores , 1974, Journal of bacteriology.

[9]  C. H. Chervenka.  Long-column meniscus depletion sedimentation equilibrium technique for the analytical ultracentrifuge. , 1970, Analytical biochemistry.

[10]  M. Zink,et al.  The distribution and substrate specificity of l-leucine dehydrogenase , 1962 .

[11]  M. Zink,et al.  L-Leucine dehydrogenase of Bacillus cereus. , 1961, Archives of biochemistry and biophysics.

[12]  O. H. Lowry,et al.  Protein measurement with the Folin phenol reagent. , 1951, The Journal of biological chemistry.