STructure of substrate-free diol dehydratase: activation mechanism of adenosylcobalamin

Noritake Yasuoka,^a Naoki Shibata,^a Jun Masuda,^a Yukio Morimoto,^a and Tetsuo Toraya^b

^aDepartment of Life Science, Himeji Institute of Technology, 3-2-1 Kouto, Kamigori, Ako-gun, Hyogo, 678-1297, Japan; ^bDepartment of Bioscience and Biotechnology, Faculty of Engineering, Okayama University, Tsushima-Naka, Okayama 700-8530, Japan (yasuoka@spring8.or.jp)

We will discuss the activation mechanism of adenosylcobalamin based on the crystal structures of diol dehydratase in the presence and absence of substrate. Diol dehydratase catalyzes the conversion of 1,2-diols to the corresponding deoxy aldehydes. The reaction requires adenosylcobalamin and a monovalent cation as cofactors. Adenosylcobalamin has a cobalt-carbon (Co-C) bond whose cleavage is required to initiate enzymatic reaction.

We have determined crystal structures of substrate-bound diol dehydratase with cyanocobalamin[1,2] and with adeninylpentylcobalamin[2]. These structures indicated the state of adenosylcobalamin after activation. To elucidate its activation mechanism upon substrate binding, the substrate-free form of the enzyme is required. We crystallized diol dehydratase in the absence of substrate and determined its crystal structure[3]. Crystals of substrate-free enzyme were obtained from the similar conditions to those previously reported except that the protein solution was prepared without substrate. Diffraction data were collected at the BL41XU beamline at SPring-8, Japan. The crystal diffracted to 1.85 Å resolution. The structure was determined by molecular replacement and refined at 1.85 Å resolution.

The b-subunit of the substrate-free form is tilted by ~2˚ and cobalamin is also tilted so that pyrrole rings A and D are significantly lifted up toward the substrate-binding site. At the substrate binding-site, two water molecules occupy the site instead of 1,2-propanediol. Based on superimposition of the substrate-free structure on the substrate-bound structure, the length between Co and C(5’) of adenosylcobalamin is estimated at 2.98 Å in the substrate-free form and 3.11 Å in the substrate-bound form. The difference of the bond length (0.13 Å) is considerably smaller than the difference between free-coenzyme (1.97 Å) and substrate-free form. Bond angles as well as bond length indicate distortion of the Co-C bond. The C(5’)-Co-N22(ring B) bond angles of the substrate-free form, the substrate-bound form and free adenosylcobalamin are 59.4˚, 46.9˚ and 84.0˚, respectively. These lines of evidences indicate that the Co-C bond is largely activated upon coenzyme binding to the apoenzyme in the absence of substrate followed by smaller activation through the substrate-induced conformational change of the enzyme.

References

1 Shibata, N., Masuda, J., Tobimatsu, T., Toraya, T., Suto, K., Morimoto, Y. and Yasuoka, N. (1999) Structure, 7, 997-1008.

2 Masuda, J., Shibata, N., Morimoto, Y., Toraya, T. and Yasuoka, N. (2000) Structure, 8, 775-788.

3 Shibata, N., Masuda, J., Morimoto, Y., Yasuoka, N. and Toraya, T. (2002) Biochemistry, 41, 12607-12617.