Crystallization and preliminary X-ray crystallographic studies of Escherichia colixanthine phosphoribosyltransferase

Abstract

Xanthine phosphoribosyltransferase (XPRT; EC 2.4.2.22) fromEscherichia coliis a purine salvage enzyme which synthesizes the nucleotides GMP, XMP, and IMP. A mutant C59A, which is more stable than wild-type XPRT while retaining high activity, has been prepared and crystallized to give three different crystal forms (A, B, and C). Form A crystals are orthorhombic (P21212), with unit cell dimensionsa= 59.2 Å,b= 92.9 Å,c= 53.2 Å. Form B crystals are monoclinic (C2) with unit cell dimensionsa= 84.4 Å,b= 70.8 Å,c= 54.1 Å, and β = 113.4°, and form C crystals are tetragonal (P41212 or P43212) with unit cell dimensionsa,b= 94 Å,c= 167.5 Å. Wild-type XPRT and a selenomethionine derivative of C59A XPRT have also been crystallized in the orthorhombic form. The selenomethionine derivative was prepared by expressing XPRT in the usualE. colistrain without the need for a methionine auxotroph. Cells were grown in a methionine-deficient medium supplemented with selenomethionine which gave >95% incorporation. Both the wild-type and selenomethionine C59A XPRT crystals are isomorphous with C59A form A crystals.