OVEREXPRESSION OF BACILLUS-SUBTILIS PHOSPHORIBOSYLPYROPHOSPHATE SYNTHETASE AND CRYSTALLIZATION AND PRELIMINARY-X-RAY CHARACTERIZATION OF THE FREE ENZYME AND ITS SUBSTRATE EFFECTOR COMPLEXES

Bacillus subtilis phosphoribosylpyrophosphate(PRPP) synthetase has bee n expressed to high levels in an Escherichia coli host strain devoid o f endogenous PRPP synthetase, A rapid and efficient purification proto col has been developed allowing production of enzyme preparations with purity conforming to the stringent criteria required for crystallizat ion. Crystallization experiments, in combination with dynamic light sc attering studies, have led to the production of three crystal forms of the enzyme, These forms include the free enzyme, the enzyme in a bina ry complex with the substrate adenosine triphosphate (ATP), and the en zyme in a quaternary complex with the substrate analog alpha, beta-met hylene adenosine triphosphate (mATP), the substrate ribose-5-phosphate (Rib-B-P), and the allosteric inhibitor adenosine diphosphate (ADP), Diffraction data showed that all three crystal forms are suitable for structure determination, They crystallize in the same hexagonal space group, P6(3), with virtually identical unit cell dimensions of a = b = 115.6 Angstrom, c = 107.8 Angstrom, and with two molecules in the asy mmetric unit, The self-rotation function showed the existence of a non -crystallographic twofold axis perpendicular to the c axis, The availa bility of the different complexes should allow questions regarding the molecular mechanisms of catalysis and allostery in PRPP synthetase to be addressed. (C) 1996 Wiley-Liss, Inc.