PURIFICATION AND CRYSTALLIZATION OF BENZOYLFORMATE DECARBOXYLASE

A new large-scale purification method for benzoylformate decarboxylase from Pseudomonas putida has allowed us to undertake an X-ray crystall ographic study of the enzyme. The previously observed instability of t he enzyme was overcome by addition of 100 mu M thiamine pyrophosphate to buffers used in the purification. The final enzyme preparation was more than 97% pure, as determined by denaturing gel electrophoresis an d densitometry. The mobility of the enzyme on a gel filtration column indicates that it is a tetramer of 57-kDa subunits. Large, single crys tals of benzoylformate decarboxylase were grown from solutions of buff ered polyethylene glycol 400, pH 8.5. The crystals diffract to beyond 1.6 Angstrom resolution and are stable for days to X-ray radiation. An alysis of X-ray data from the crystals, along with the newly determine d quaternary structure, identifies the space group as 1222. The unit c ell dimensions are a = 82 Angstrom, b = 97 Angstrom, c = 138 Angstrom. An average V-m value for the crystals is consistent with one subunit per asymmetric unit. The subunits of the tetramer must be arranged wit h tetrahedral 222 symmetry.