Browse > Article

Stabilization of Quinonoid Intermediate E-Q by Glu32 of D-Amino Acid Transaminase  

Ro Hyeon-Su (Department of Microbiology and Research Institute of Life Science, Gyeongsang National University)
Jeon Che-Ok (Environmental Biotechnology National Core Research Center, Gyeongsang National University)
Kim Hak-Sung (Department of Biological Sciences, Korea Advanced Institute of Science and Technology)
Sung Moon-Hee (Department of Bio- and Nanochemistry, Kookmin University)
Publication Information
Journal of Microbiology and Biotechnology / v.16, no.9, 2006 , pp. 1434-1440 More about this Journal
Abstract
The stable anchorage of pyridoxal 5'-phosphate (PLP) in the active site of D-amino acid transaminase (D-AT) is crucial for the enzyme catalysis. The three-dimensional structure of D-AT revealed that Glu32 is one of the active site groups that may playa role in PLP binding. To prove the role of Glu32 in PLP stability, we firstly checked the rate of the potential rate-limiting step. The kinetic analysis showed that the rate of the ${\alpha}$-deprotonation step reduced to 26-folds in E32A mutant enzyme. Spectral analyses of the reaction of D-AT with D-serine revealed that the E32A mutant enzyme failed to stabilize the key enzyme-substrate intermediate, namely a quinonoid intermediate (E-Q). Finally, analysis of circular dichroism (CD) on the wild-type and E32A mutant enzymes showed that the optical activity of PLP in the enzyme active site was lost by the removal of the carboxylic group, proving that Glu32 is indeed involved in the cofactor anchorage. The results suggested that the electrostatic interaction network through the groups from PLP, Glu32, His47, and Arg50, which was observed from the three-dimensional structure of the enzyme, plays a crucial role in the stable anchorage of the cofactor to give necessary torsion to the plane of the cofactor-substrate complex.
Keywords
Transaminase; quinonoid; cofactor; pyridoxal; D-amino acid;
Citations & Related Records
Times Cited By KSCI : 4  (Citation Analysis)
Times Cited By Web Of Science : 0  (Related Records In Web of Science)
연도 인용수 순위
1 Bae, H. S., S.-P. Hong, S.-G. Lee, M.-S. Kwak, N. Esaki, and M.-H. Sung. 2002. Application of a thermostable glutamate racemase from Bacillus sp. SK-1 for the production of d-phenylalanine in a multi-enzyme system. J. Mol. Catal. B-Enzym. 17: 223-233   DOI   ScienceOn
2 Hayashi, H. and H. Kagamiyama. 1995. Reaction of aspartate aminotransferase with L-erythro-3-hydroxyaspartate: Involvement of Tyr70 in stabilization of the catalytic intermediate. Biochemistry 34: 9413-9423   DOI   ScienceOn
3 Jensen, R. A. and W. Gu. 1996. Evolutionary recruitment of biochemically specialized subdivisions of family I within the protein superfamily of aminotransferases. J. Bacteriol. 178: 2161-2171   DOI
4 Futaki, S., H. Ueno, A. Martinez del Pozo, M. A. Pospischil, J. M. Manning, D. Ringe, B. Stoddard, K. Tanizawa, T Yoshimura, and K. Soda. 1990. Substitution of glutamine for lysine at the pyridoxal phosphate binding site of bacterial d-amino acid transaminase. J. Biol. Chem. 265: 22306-22312
5 Lee, S. G., S. P. Hong, J. J. Song, S. J. Kim, M. S. Kwak, and M. H. Sung. 2006. Functional and structural characterization of thermostable D-amino acid aminotransferases from Geobacillus spp. Appl. Environ. Microbiol. 72: 1588-1594   DOI   ScienceOn
6 Yonaha, K., H. Misono, T. Yamamoto, and K. Soda. 1975. D-Amino acid aminotransferase of Bacillus sphaericus. Enzymologic and spectrometric properties. J. Biol. Chem. 250: 6983-6989
7 Park, H., K. S. Lee, S. M. Park, K. W. Lee, A. Y. Kim, and Y. M. Chi. 2005. Relationship between enhancement of electrostriction and decrease of activation energy in porcine pancreatic lipase catalysis. J. Microbiol. Biotechnol. 15: 587-594   과학기술학회마을
8 Soper, T. S. and J. M. Manning. 1985. Enzyme-activated inhibitors of pyridoxal-phosphate enzymes, pp. 266-285. In Christen, P. and D. E. Metzler (eds.), Transaminases. John Wiley and Sons, New York, U.S.A
9 Gallagher, D. T, G. L. Gilliland, G. Xiao, J. Zondlo, K. E. Fisher, D. Chinchilla, and E. Eisenstein. 1998. Structure and control of pyridoxal phosphate dependent allosteric threonine deaminase. Structure 6: 465-475   DOI   ScienceOn
10 Jung, B. R., Y. Lee, Y. Lim, and J. H. Ahn. 2005. Structure prediction of the peptide synthesized with the nonribosomal peptide synthetase gene from Bradyrhizobium japonicum. J. Microbiol. Biotechnol. 15: 656-659   과학기술학회마을
11 Morino, Y. and S. Tanase. 1985. Quasisubstrates and irreversible inhibitors of aspartate aminotransferase, pp. 251-265. In Christen, P. and D. E. Metzler (eds.), Transaminases. John Wiley and Sons, New York, U.S.A
12 Jung, H. J., K. S. Choi, and D. G. Lee. 2005. Synergistic killing effect of synthetic peptide P20 and cefotaxime on methicillin-resistant nosocomial isolates of Staphylococcus aureus. J. Microbiol. Biotechnol. 15: 1039-1046   과학기술학회마을
13 Kim, J. H., J. J. Song, B. G. Kim, M. H. Sung, and S. C. Lee. 2004. Enhanced stability of tyrosine phenol-lyase from Symbiobacterium toebii by DNA shuffling. J. Microbiol. Biotechnol. 14: 153-157
14 Ro, H. S., S. P. Hong, T. Yoshimura, N. Esaki, K. Soda, H. S. Kim, and M. H. Sung. 1996. Site-directed mutagenesis of the amino acid residues in $\beta$-strand III of D-amino acid aminotransferase. FEBS Lett. 398: 141 -145   DOI   ScienceOn
15 Sugio, S., G. A. Petsko, J. M. Manning, K. Soda, and D. Ringe. 1995. Crystal structure of a D-amino acid aminotransferase: How the protein controls stereoselectivity. Biochemistry 34: 9661-9669   DOI   ScienceOn
16 Rhee, S., K. D. Parris, C. C. Hyde, S. A. Ahmed, E. W. Miles, and D. R. Davies. 1997. Crystal structures of a mutant (${\beta}$-K87T) tryptophan synthase ${\alpha}2{\beta}2$ complex with ligands bound to the active sites of the ${\alpha}$- and ${\beta}$-subunits reveal ligand-induced conformational changes. Biochemistry 36: 7664-7680   DOI   ScienceOn
17 Burkhard, P., G. S. Rao, E. Hohenester, K. D. Schnackerz, P. F. Cook, and J. N. Jansonius. 1998. Three-dimensional structure of O-acetylserine sulfhydrylase from Salmonella typhimurium. J. Mol. Biol. 283: 121-133   DOI   ScienceOn
18 Toney, M. D., E. Hohenester, J. W. Keller, and J. N. Jansonius. 1995. Structural and mechanistic analysis of two refined crystal structures of the pyridoxal phosphate-dependent enzyme dialkylglycine decarboxylase. J. Mol. Biol. 245: 151-179   DOI   ScienceOn
19 Hyde, C. C., S. A. Ahmed, E. A. Padlan, E. W. Miles, and D. R. Davies. 1988. Three-dimensional structure of the tryptophan synthase ${\alpha}2{\beta}2$ multienzyme complex from Salmonella typhimurium. J. Biol. Chem. 263: 17857-17871
20 Martinez del Pozo, A., M. A. Pospischill, H. Ueno, J. M. Manning, K. Tanizawa, K. Nishimura, K. Soda, D. Ringe, B. Stoddard, and G. A. Petsko. 1989. Effects of D-serine on bacterial D-amino acid aminotransferase: Accumulation of an intermediate and inactivation of the enzyme. Biochemistry 28: 8798-8803   DOI   ScienceOn
21 Lee, S. G. and M. H. Sung. 1999. Development of an enzymatic system for the production of dopamine from catechol, pyruvate, and ammonia. Enz. Microb. Technol. 25: 298-302   DOI   ScienceOn
22 Ro, H. S. and E. W. Miles. 1999. Structure and function of the tryptophan synthase ${\alpha}2{\beta}2$ complex: Roles of ${\beta}$ subunit histidine 86. J. Biol. Chem. 274: 36439-36445   DOI   ScienceOn