J. Biol. Chem., Vol. 262, Issue 32, 15323-15326, Nov, 1987
X-ray crystallographic studies of the alanine-specific racemase from Bacillus stearothermophilus. Overproduction, crystallization, and preliminary characterization
DJ Neidhart, MD Distefano, K Tanizawa, K Soda, CT Walsh and GA Petsko
Department of Chemistry, Massachusetts Institute of Technology, Cambridge 02139.
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.
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