X-ray crystallographic studies of the alanine-specific racemase from Bacillus stearothermophilus. Overproduction, crystallization, and preliminary characterization.
Academic Article
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abstract
To facilitate large-scale purification and crystallographic study, we have subcloned the gene for the alanine racemase of Bacillus stearothermophilus from pICR401 (Inagaki, K., Tanizawa, K., Badet, B., Walsh, C. T., Tanaka, H., and Soda, K. (1986) Biochemistry 25, 3268-3274) and overproduced the enzyme in Escherichia coli W3110 lacIq using the tac promoter of PKK223-3. This system yields alanine racemase as 6% of the bacterial cytosolic protein. Purification by a modification of the procedure of Inagake et al. yielded 75 mg of homogeneous alanine racemase from 30 g of cells (wet weight). Large, well-formed crystals of alanine racemase have been grown from polyethylene glycol 8000 using vapor diffusion. These crystals have unit cell dimensions a = 85.3 A, b = 110.0 A, and c = 89.9 A. The crystals belong to space group P2(1), with beta fortuitously equal to 90 degrees within experimental error; however, they are frequently twinned by second order pseudomerohedry with twin fraction (the ratio of the volume of the smaller twin domain to the total volume of the crystal) ranging from about 0 to 0.5. Fortunately, for crystals with low twin fraction, computational methods have been developed for the analysis and correction of simple twinning (Fisher, R. G., and Sweet, R. M. (1980) Acta Crystallogr. A36, 755-760). The crystals contain two alpha 2 dimers of alanine racemase in the asymmetric unit. We have identified several potentially useful heavy atom derivatives in low resolution screening experiments and are proceeding with high resolution data collection.
