PRELIMINARY CRYSTALLOGRAPHIC STUDY OF FORMALDEHYDE DISMUTASE. Akihito Yamano¹, Tsuneyuki Higashi¹, Hideshi Yanase², Nobuo Kato³, Hideaki Moriyama⁴, Nobuo Tanaka⁴ & Yukiteru Katsube¹, ¹X-ray Research Laboratory, Rigaku Corp., Tokyo, Japan, ²Department of Biotechnology, Faculty of Engineering, Tottori University, Tottori, Japan, ³Department of Agricultural Chemistry, Faculty of Agriculture, Kyoto University, Kyoto, Japan, ⁴Department of Life Science, Faculty of Bioscience and Biotechnology, Tokyo Institute of Technology, Kanagawa, Japan.

Formaldehyde dismutase (FDM) (MW=180kDa) was crystallized by the hanging drop vapor diffusion technique. FDM, found in a formaldehyde resistant bacterium (Pseudomonas putida), catalyzes the dismutation of aldehydes and alcohol. Amino acid sequence alignment indicates that FDM has the NAD-binding motif conserved among the NAD-dependent dehydrogenases. However, the binding mode of NAD(H) in FDM appears to be very different from those in other NAD-dependent dehydrogenases. The NAD(H) in FDM does not dissociate but stays bound in the enzyme throughout the reaction, therefore there is no enhancement of its activity on the addition of an excess amount of NAD(H). In order to elucidate the nature of the stronger binding, we started a project to determine the three-dimensional structure of FDM.

Crystals suitable for an X-ray diffraction experiment were acquired by equilibration of FDM solution (17.5 mg/ml) against 30%(wt/v) AS in 100mM potassium phosphate buffer (pH7.0). The protein solution was filtered to eliminate unwanted aggregation detected by a DynaPro (Protein Solutions) prior to the crystallization. FDM crystallized in the tetragonal space group P4₁ (or P4₃) with unit cell dimensions a=b=92.4Å and c=225.0Å. X-ray diffraction data were collected to 3Å resolution on an RAXIS IIc. We are now trying MR methods using coordinates from NAD-dependent dehydrogenase structures.