Crystallization and aldo-keto reductase activity of Gcy1p from Saccharomyces cerevisiae

Crystallization and preliminary X-ray diffraction studies of Gcy1p, an aldo-keto reductase from Saccharomyces cerevisiae, have been performed. Both the wild type and a double-mutant form of Gcy1p were crystallized using the hanging-drop method at 298 K; however, only the double-mutant form has so far yielded crystals suitable for X-ray diffraction analysis. These crystals belonged to the primitive monoclinic space group P2(1), with unit-cell parameters a = 50.94, b = 65.64, c = 86.23 Angstrom, beta = 92.64 degrees. Diffraction data were collected to 2.5 Angstrom. Assuming two 35 kDa subunits in the asymmetric unit yielded a V-m of 2.06 Angstrom(3) Da(-1). Additionally, a kinetic study performed by measuring the rate of oxidation of NADPH in the presence of several substrates indicates that both wild-type and double-mutant proteins are enzymes possessing NADPH-dependent reductase activity.