1. Current Research and Principal Research Interests
My research is concerned with the three-dimensional structures of proteins and their biochemical functions. To this end, research is focussed on modification of the catalytic properties of a number of pharmaceutically or industrially important enzymes. The tools used by X-ray crystallography, design of transition-state analog inhibitors, and site-directed mutagenesis. Our aims are to learn how an enzyme obtains their reaction specificity and substrate selectivity and to dissect the catalytic mechanism.
Pyridoxal 5'-phosphate (vitamin B6) is capable of catalyzing several types of transformations. However, only one enzyme catalyzes for single type of transformations with a substrate specificity. We are studying a number of these enzymes structurally in order to determine how the protein controls the chemical outcome of such transformation
Aspartate aminotransferase(AAT) is one of the best-studied vitamin B6 dependent enzymes. Systematic single and multiple replacement studies have been applied to Escherichia coli AAT, and the role of active site residues or the catalytic mechanism has been proposed from X-ray crystallographic and enzymological studies. Recently, the structures of native histidinol-phosphate aminotransferase (HspAT) and its substrate complexes have been solved. The HspAT recognizes substrates by utilizing essentially the same active-site folding as that of AAT, conserving the essential residues for transamination reaction, and replacing and relocating some of the active-site residues.
Based on the similarities in sequence, many PLP-dependent enzymes are placed in fold type I, while 4-amino-4-deoxychorismate lyase (ADCL) is placed in fold type IV together with D-amino acid aminotransferase (DAAT) and branched-chain amino acid aminotransferase (BCAT). Although the main chain folding of the active site active site is homologous to those of DAAT and BCAT, no longer in active site are conserved among them except for the residues which directly interact with the coenzyme and play critical roles in the catalytic functions.
2. Selected Publications
1. “Three-dimensional Structure of the Flavoenzyme Acyl-CoA Oxidase-II from Rat Liver, the Peroxisomal Counterpart of Mitochondrial Acyl-CoA Dehydrogenase”, Y. Nakajima, I. Miyahara, K. Hirotsu, Y. Nishina, K. Shiga, C. Setoyama, H. Tamaoki, and R. Miura, J. Biochem., 131, 365-374 (2002).
2. “Effects of hydrogen bonds in association with flavin and substrate in flavoenzyme D-amino acid oxidase. The catalytic and structural roles of Gly313 and Thr317”, C. Setoyama, Y. Nishina, H. Tamaoki, H. Mizutani, I. Miyahara, K. Hirotsu, K. Shiga, and R. Miura, J. Biochem., 131, 59-69 (2002).
3. “Crystal Structure of Quinohemoprotein Amine Dehydrogenase from Pseudomonas putida, Identification of a novel quinone cofactor encaged by multiple thio-ether cross-bridges”, A. Satoh, J-K. Kim, I. Miyahara, B. Devreese, I. Vandenberghe, A. Hacisalihoglu, T. Okajima, S. Kuroda, O. Adachi, J.A. Duine, J. Beeumen, K. Tanizawa, and K. Hirotsu, J. Biol. Chem., 277(4), 2830-2834 (2002).
4. “Substrate recognition mechanism of thermophilic dual-substrate enzyme”, H. Ura , T. Nakai, Si. Kawaguchi , I. Miyahara, K. Hirotsu, and S. Kuramitsu. J. Biochem., 130, 89-98 (2001).
5. “Porcine kidney D-amino acid oxidase: The three-dimensional structure and its catalytic mechanism based on the enzyme-substrate complex model”, R. Miura, C. Setoyama, Y. Nishina, K. Shiga, I. Miyahara, H. Mizutani, and K. Hirotsu, J. Mol. Catal. B, Enzym. 12, 43-52 (2001).
6. “Structures of Escherichia coli Histidinol-Phosphate Aminotransferase and Its Complexes with Histidinol-Phosphate and N-(5’-Phosphopyridoxyl)-L-Glutamate: Double Substrate Recognition of the Enzyme”, K. Haruyama, T. Nakai, I. Miyahara, K. Hirotsu, H. Mizuguchi, H. Hayashi, and H. Kagamiyama, Biochemistry, 40, 4633-4644 (2001).
7. “Three-Dimensional Structure of the Purple Intermediate of Porcine Kidney D-Amino Acid Oxidase. Optimization of the Oxidative Half-Reaction through Alignment of the Product with Reduced Flavin”, H. Mizutani, I. Miyahara, K. Hirotsu, Y. Nishina, K. Shiga, C. Setoyama, and R. Miura, J. Biochem. 128, 73-81 (2000).
8. “Three-Dimensional Structure of 4-Amino-4-deoxychorismate Lyase from Escherichia coli”, T. Nakai, H. Mizutani, I. Miyahara, K. Hirotsu, S. Takeda, K.-H. Jhee, T. Yoshimura, and N. Esaki, J. Biochem. 128, 29-38 (2000).
9. “Strain Is More Important Than Electrostatic Interaction in Controlling the pKa of the Catalytic Group in Aspartate Aminotransferase”, H. Mizuguchi, H. Hayashi, K. Okada, I. Miyahara, K. Hirotsu, and H. Kagamiyama, Biochemistry, 40, 353-360 (2001).
10. “Structure of Thermus thermophilus HB8 Aspartate Aminotransferase and Its Complex with Maleate”, T. Nakai, K. Okada, S. Akutsu, I. Miyahara, S. Kawaguchi, R. Kato, S. Kuramitsu and K. Hirotsu, Biochemistry, 38, 2413-2424 (1999).
|
