CRYOCRYSTALLOGRAPHY OF 3-ISOPROPYLMALATE DEHYDROGENASES AT 100 AND 150K. Nobuo Tanaka, Chikahiro Nagata, Hideaki Moriyama, Tairo Oshima, Masayoshi Nakasako²⁾, Masaki Yamamoto²⁾, Tatzuo Ueki²⁾. Faculty of Bioscience and Biotechnology, Tokyo Institute of Technology, Yokohama 226, Japan; ²⁾The Institute of Physical and Chemical Research, Wako, 351-01

In the pathway of biosynthesis of leucine, 3-isopropylmalate dehydrogenase (IPMDH) catalyses the conversion from 3-isopropylmalate to 3-ketocaproate through the dehydrogenation and decarboxylation. It was found that its chimeric enzymes between Thermus thermophilus and Bacillus subtilis show the intermediate thermostability between the parental enzymes. Their detailed structures will be quite helpful to understand the thermostability of the enzyme.

In the present study, we performed the cryo-crystallographic structure analysis on the IPMDH isolated from Thermus thermophilus, 10T, and its thermostable chimeric mutant, 2T2M6T-S82R, at 100 and at 150K, to observe the thermal behavior with the suppressed temperature factor. The diffraction data was collected from the cryo-cooled crystals on the R-AXIS IIc according to the reported method (1).

Positional deviations of the domains were detected in the subunit, though the overall structures of both enzymes were similar to the structures at room temperature. Observed shrinkage of the molecules, that calculated from the atomic coordinates, was found larger in the chimera than in the 10T. The overall temperature factors were almost the same in the structures at 100K and at 150K, although those decreased very much from that of the room temperature. The 2T2M6T-S82R showed the overall temperature factor decreased montonically with the temperature. The extrapolation of temperature factors at higher temperature indicated that the chimera vibrates more than the 10T, which may be a cause of temperature sensitivity of the chimera molecule.

1. Nakasako, M., Ueki, T., Toyoshima, C., and Umeda, Y. (1995) J. Appl. Cryst. 28, 857.