Crystal Structure of L-Lysine Dehydrogenase
(Fig. 9A). Aside from Glu168, there are no other obvious resi-
dues in the active site that might facilitate the proton transfer
expected to occur during catalysis. For that reason, we con-
structed E168Q and E168A mutants. Notably, however, the
kinetic analysis of the two mutants indicated that their catalytic
activities were nearly the same as that of the wild-type enzyme,
although the Km values for L-lysine were slightly increased to
2.0–2.3 mM. In addition, Glu168 is conserved as Glu174 in
A. tumefaciens LysDH but is replaced by His181 in G. stearo-
thermophilus LysDH (Fig. 7). Apparently, Glu168 is not essential
for enzyme catalysis. Among the residues located within the
substrate-binding pocket, Tyr156 and Arg226 are strictly con-
served in LysDHs and saccharopine reductase. We therefore
also constructed Y156F and R226K mutants and observed that
8. Misono, H., Hashimoto, H., Uehigashi, H., Nagata, S., and Nagasaki, S.
(1989) J. Biochem. 105, 1002–1008
9. Hashimoto, H., Misono, H., Nagata, S., and Nagasaki, S. (1990) Agric. Biol.
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11. Hummel, W., and Kula, M. R. (1989) Eur. J. Biochem. 184, 1–13
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17. Bradford, M. M. (1976) Anal. Biochem. 72, 248–254
these mutations completely abolished the activity of the 18. Kusakabe, H., Kodama, K., Kuninaka, A., Yoshino, H., Misono, H., and
enzyme. In our model, the side chain hydroxyl group of Tyr156
Soda, K. (1980) J. Biol. Chem. 255, 976–981
and the guanidium group of Arg226 (NH2) are estimated to be
19. Ohshima, T., Misono, H., and Soda, K. (1978) J. Biol. Chem. 253,
5719–5725
7.8 Å from C-6 of L-lysine, whereas the corresponding groups in
20. Otwinowski, Z., and Minor, W. (1997) Methods Enzymol. 276, 307–326
Tyr170 and Arg247 (NH1) of saccharopine reductase are 7.1–7.5
21. Terwilliger, T. C., and Berendzen, J. (1999) Acta Crystallogr. Sect. D 55,
Å from C-6 of saccharopine. These distances preclude the
involvement of these two residues in the hydride transfer.
Instead, it appears that Tyr156 and Arg226 play essential roles in
the binding of substrates to LysDHs.
849–861
22. Terwilliger, T. C. (2000) Acta Crystallogr. Sect. D 56, 965–972
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Sect. D 53, 240–255
Although the mechanism underlying the catalytic reaction of
P. horikoshii LysDH remains unclear, this study provides the
first structural insight into substrate stereo recognition and
hydride transfer by LysDH. The structure of the P. horikoshii
LysDHꢀNADꢀsubstrate analog ternary complex should be a use-
ful focus for further investigation.
25. Bru¨nger, A. T., Adams, P. D., Clore, G. M., DeLano, W. L., Gros, P.,
Grosse-Kunstleve, R. W., Jiang, J. S., Kuszewski, J., Nilges, M., Pannu, N. S.,
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Acknowledgments—We thank Drs. N. Igarashi, N. Matsugaki, and S.
Wakatsuki for help in data collection at Photon Factory beamline
BL5A. We also thank Drs. S. Kawamura, T. Araki, and T. Torikata for
extremely helpful support.
28. Potterton, L., McNicholas, S., Krissinel, E., Gruber, J., Cowtan, K., Emsley,
P., Murshudov, G. N., Cohen, S., Perrakis, A., and Noble, M. (2004) Acta
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MARCH 12, 2010•VOLUME 285•NUMBER 11
JOURNAL OF BIOLOGICAL CHEMISTRY 8453