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Identification of Essential Amino acid Residues in Valine Dehydrogenase from Streptomyces albus  

Hyun Chang-Gu (Division of Bioscience & Bioinformatics, Myongji University)
Kim Sang-Suk (Division of Bioscience & Bioinformatics, Myongji University)
Suh Joo-Won (Division of Bioscience & Bioinformatics, Myongji University)
Publication Information
Journal of Microbiology / v.44, no.1, 2006 , pp. 50-53 More about this Journal
Abstract
Cys-29 and Cys-251 of Streptomyces albus valine dehydrogenase(ValDH) were highly conserved in the corresponding region of $NAD(P)^+$-dependent amino acid dehydroganase sequences. To ascertain the functional role of these cysteine residues in S. albus ValDH, site-directed mutagenesis was performed to change each of the two residues to serine. Kinetic analyses of the enzymes mutated at Cys-29 and Cys-251 revealed that these residues are involved in catalysis. We also constructed mutant ValDH by substituting valine for leucine at 305 by site-directed mutagenesis. This residue was chosen, because it has been proposed to be important for substrate discrimination by phenylalanine dehydrogenase (PheDH) and leucine dehydrogenase (LeuDH). Kinetic analysis of the V305L mutant enzyme revealed that it is involved in the substrate binding site. However it displayed less activity than the wild type enzyme toward all aliphatic and aromatic amino acids tested.
Keywords
Streptomyces; valine dehydrogenase; metagenesis; kinetic analysis;
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1 Kopp, J. and T. Schwede. 2004. The SWISS-MODEL Repository of annotated three-dimensional protein structure homology models. Nucleic Acids Res. 32, D230-D234   DOI
2 Ohshima, T., N. Nishida, S. Bakthavatsalam, K. Kataoka, H. Takada, T. Yoshimura, N. Esaki and K. Soda. 1994. The purification, characterization, cloning and sequencing of the gene for a halostable and thermostable leucine dehydrogenase from Thermoactinomyces intermedius. Eur. J. Biochem. 222, 305-312   DOI   ScienceOn
3 Seah, S.Y., K.L. Britton, D.W. Rice, Y. Asano and P.C. Engel. 2002. Single amino acid substitution in Bacillus sphaericus phenylalanine dehydrogenase dramatically increases its discrimination between phenylalanine and tyrosine substrates. Biochemistry 41, 11390-11397   DOI   ScienceOn
4 Turnbull, A.P., P.J. Baker and D.W. Rice. 1997a. Analysis of the quaternary structure, substrate specificity, and catalytic mechanism of valine dehydrogenase. J. Biol. Chem. 272, 25105-25111   DOI   ScienceOn
5 Hyun, C.G., S.S. Kim, I.H. Lee and J.W. Suh. 2000a. Alteration of substrate specificity of valine dehydrogenase from Streptomyces albus. Antonie Van Leeuwenhoek. 78, 237-242   DOI   ScienceOn
6 Baker, P.J., A.P. Turnbull, S.E. Sedelnikova, T.J. Stillman and D.W. Rice. 1995. A role for quaternary structure in the substrate specificity of leucine dehydrogenase. Structure 3, 693-705   DOI   ScienceOn
7 Turnbull, A.P., S.R. Ashford, P.J. Baker, D.W. Rice, F.H. Rodgers, T.J. Stillman, and R.L. Hanson. 1994b. Crystallization and quaternary structure analysis of the NAD(+)-dependent leucine dehydrogenase from Bacillus sphaericus. J. Mol. Biol. 236, 663-665   DOI   ScienceOn
8 Bradford, M.M. 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   DOI   ScienceOn
9 Nguyen, L.T., K.T. Nguyen, J. Spizek and V. Behal. 1995. The tylosin producer, Streptomyces fradiae, contains a second valine dehydrogenase. Microbiology 141, 1139-1145   DOI   ScienceOn
10 Seah, S.Y., K.L. Britton, D.W. Rice, Y. Asano and P.C. Engel. 2003. Kinetic analysis of phenylalanine dehydrogenase mutants designed for aliphatic amino acid dehydrogenase activity with guidance from homology-based modelling. Eur. J. Biochem. 270, 4628-4634   DOI   ScienceOn
11 Nagata, S., K. Tanizawa, N. Esaki, Y. Sakamoto, T. Ohshima, H. Tanaka and K. Soda. 1988. Gene cloning and sequence determination of leucine dehydrogenase from Bacillus stearothermophilus and structural comparison with other $NAD(P)^+$ dependent dehydrogenases. Biochemistry 27, 9056-9062   DOI   ScienceOn
12 Omura, S., K. Tsuzuki, Y. Tanaka, H. Sakakibara, M. Aizawa and G. Lukacs. 1983. Valine as a precursor of n-butyrate unit in the biosynthesis of macrolide aglycone. J. Antibiot. 36, 614-616
13 Seah, S.Y., K.L. Britton, P.J. Baker, D.W. Rice, Y. Asano and P.C. Engel. 1995. Alteration in relative activities of phenylalanine dehydrogenase towards different substrates by site-directed mutagenesis. FEBS Lett. 370, 93-96   DOI   ScienceOn
14 Oikawa, T., K. Yamanaka, T. Kazuoka, N. Kanzawa and K. Soda. 2001. Psychrophilic valine dehydrogenase of the antarctic psychrophile, Cytophaga sp. KUC-1: purification, molecular characterization and expression. Eur. J. Biochem. 268, 4375-4383   DOI   ScienceOn
15 Sherman, M.M., S. Yue and C.R. Hutchinson. 1986. Biosynthesis of lasalocid A. Metabolic interrelationships of carboxylic acid precursors and polyether antibiotics. J. Antibiot. 39, 1135-1143   DOI
16 Hyun, C.G., S.S Kim, K.H. Park and J.W. Suh. 2000b. Valine dehydrogenase from Streptomyces albus: gene cloning, heterologous expression and identification of active site by site-directed mutagenesis. FEMS Microbiol. Lett. 182, 29-34   DOI