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 been expressed to high levels in an Escherichia coli host strain devoid of endogenous PRPP synthetase. A rapid and efficient purification protocol has been developed allowing production of enzyme preparations with purity conforming to the stringent criteria required for crystallization. Crystallization experiments, in combination with dynamic light scattering studies, have led to the production of three crystal forms of the enzyme. These forms include the free enzyme, the enzyme in a binary complex with the substrate adenosine triphosphate (ATP), and the enzyme in a quaternary complex with the substrate analog alpha, beta-methylene adenosine triphosphate (mATP), the substrate ribose-5-phosphate (Rib-5-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 angstroms, c = 107.8 angstrom, and with two molecules in the asymmetric unit. The self-rotation function showed the existence of a non-crystallographic twofold axis perpendicular to the c axis. The availability of the different complexes should allow questions regarding the molecular mechanisms of catalysis and allostery in PRPP synthetase to be addressed.
Bentsen
A K
AK
Center for Enzyme Research, Department of Protein Chemistry, University of Copenhagen, Denmark.
Larsen
T A
TA
Kadziola
A
A
Larsen
S
S
Harlow
K W
KW
D000255:Adenosine Triphosphate / Q000737:chemistry; D001412:Bacillus subtilis / Q000201:enzymology* / Q000235:genetics; D018360:Crystallography, X-Ray; D004926:Escherichia coli / Q000235:genetics; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry; D012268:Ribose-Phosphate Pyrophosphokinase / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics; D012267:Ribosemonophosphates / Q000737:chemistry
D011994:Recombinant Proteins; D012267:Ribosemonophosphates; C031626:ribose-5-phosphate; D000255:Adenosine Triphosphate; D012268:Ribose-Phosphate Pyrophosphokinase