Laboratory for Biological Structural Chemistry:
Structure-Function Relationship of Enzymes and Protein
Crystallography

http://www.sci.osaka-cu.ac.jp/chem/chem.html

Current Research and Principal Research Interests
Selected Publications

1. Current Research and Principal Research Interests
We are engaged in structure analysis of proteins by X-ray crystallography. The overall aim ofour research is to learn the relationship of the three-dimensional structure of protein to its
biochemical function and the basic concept of the molecular design. One area of interest is themechanism of the induced fit and substrate recognition of proteins. A second is to understandthe electron and proton transfer among protein, cofactor and substrate through the catalytic
process. A third area of interest is the structural and evolutionary relationship between andwithin families of proteins. The tools used are purification, crystallization, X-ray analysis,computer graphics and molecular biology (especially protein expression and site-directedmutagenesis). A strong X-ray source given by a third generation synchrotron facility (SPring-8)
and a focused X-ray from in- house equipment with a two-dimensional detector haveparticularly raised the level of macromolecular crystallography. Now we can determine not only
the precise structure of a protein, but also the structure of its reaction intermediate. A series ofproteins related to one another such as a family of vitamin B6 dependent enzymes and all
proteins coded on a structural gene can be subjected to X-ray crystallographic studies.
For a long time, we had been working on small molecule crystallography. We then began
macromolecular crystallography as part of our research activity, and about three years ago
shifted almost completely to this area.

aspartate aminotransferase

branched-chain amino acid aminotransferase

For many years we have worked on the structure-function relationship in a series of pyridoxal
5'-phosphate (vitamin B6) dependent enzymes. Vitamin B6 enzymes play important roles in theamino acid metabolism, and catalyze a wide range of amino acid transformation(transamination, decarboxylation, deamination, racemization and aldol cleavages) by labilizing
one of three bonds at C carbon atom of an amino acid substrate. Importantly, one enzyme
usually catalyzes one reaction with a substrate specificity. The vitamin B6 dependent enzymes
are classified into fold type I-IV, indicating that the enzymes evolved from four common
ancestors, and the versatility of enzymes has been attained by divergent and convergent
molecular evolution. The aim of this work is to uncover the underlying mechanism in the
catalytic events of vitamin B6 enzymes, and to understand the action of B6 enzymes in general.
The structures of many aminotransferases such as aspartate, branched-chain, histidinol
phosphate, and aromatic amino acid aminotranferases have been determined to provide someinsight into the substrate recognition, the role of the active site residues, the reaction mechanism,and the evolutionary relationship of the vitamin B6 dependent enzymes. In addition totransaminase, we are studying a number of vitamin B6 enzymes such as decarboxylase,
deaminase, lyase and racemase.

We are also interested in the catalytic action offlavin (vitamin B2) dependent enzymes.
D-Amino acid oxidase catalyzes the oxidative
deamination of D-amino acids. (DAO) Although
DAO has been one of the most extensivelyinvestigated flavoenzymes, the catalytic
mechanism of DAO was a matter yet to besettled. X-ray structure of DAO revealed that
there was no presumed protein base to abstract
the -proton of a substrate amino acid, allowing us
to propose new catalytic mechanisms on theactive-site model of enzyme-substrate complex.
Two possible mechanisms for the reductive-halfreaction are the electron-proton-electron transfer
mechanism and the ionic mechanism. The
structure of the purple intermediate of DAO,
which is a complex between the dehydrogenatedD-amino acid oxidase
product and the reduced DAO, was solved bycryo-X-ray crystallography. It was shown that the oxidative half-reaction of DAO is optimized
through the alignment of the product with respect to reduced flavin. The X-ray studies onanother flavoenzymes are ongoing.
A new and exciting project involves a quinoprotein that has a presumed quinone cofactor, andtwo hemes as redox active group. The X-ray structure of quinohemo-

Novel cofactor (cysteine tryptophylquinone ) and hitherto unknown
cross-links
Figure 3 Two-photon reaction via hot benzene produce 1,3-hexadiene-5-yne as a dominantphotoproduct. The corresponding vibrational temperature of benzene with two-photon energy ofArF laser light is evaluated to be as high as 5900 K.

2. Selected Publications
1. “Crystal Structure of Argininosuccinate Synthetase from Thermus thermophilus HB8:
Structural Basis for the Catalytic Action”, M. Goto, Y. Nakajima, and K. Hirotsu, J. Biol.
Chem., 277, 15890-15896 (2002).

2. “Three-dimensional Structure of the Flavoenzyme Acyl-CoA Oxidase-II from Rat Liver, thePeroxisomal 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).

3. “Effects of hydrogen bonds in association with flavin and substrate in flavoenzyme D-aminoacid 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).

4. “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).

5. “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).

6. “Structures of Escherichia coli Branched-Chain Amino Acid Aminotransferase and Its
Complexes with 4-Methylvalerate and 2-Methylleucine: Induced Fit and Substrate Recognitionof the Enzyme”, K. Okada, K. Hirotsu, H. Hayashi, and H. Kagamiyama, Biochemistry, 40,7453-7463 (2001).

7. “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).

8. “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).

9. “Three-Dimensional Structure of the Purple Intermediate of Porcine Kidney D-Amino AcidOxidase. Optimization of the Oxidative Half-Reaction through Alignment of the Product withReduced Flavin”, H. Mizutani, I. Miyahara, K. Hirotsu, Y. Nishina, K. Shiga, C. Setoyama, andR. Miura, J. Biochem., 128, 73-81 (2000).

10. “Three-Dimensional Structure of 4-Amino-4-deoxychorismate Lyase from Escherichiacoli”, T. Nakai, H. Mizutani, I. Miyahara, K. Hirotsu, S. Takeda, K.-H. Jhee, T. Yoshimura, andN. Esaki, J. Biochem., 128, 29-38 (2000).