d-Ribose-5-Phosphate Isomerase from Spinach: Heterologous Overexpression, Purification, Characterization, and Site-Directed Mutagenesis of the Recombinant Enzyme

A cDNA encoding spinach chloroplastic ribose-5-phosphate isomerase (RPI) was cloned and overexpressed in Escherichia coli, and a purification scheme for the recombinant enzyme was developed. The purified recombinant RPI is a homodimer of 25-kDa subunits and shows kinetic properties similar to those of the homodimeric enzyme isolated from spinach leaves (A. C. Rutner, 1970, Biochemistry 9, 178–184). Phosphate, used as a buffer in previous studies, is a competitive inhibitor of RPI with a Ki of 7.9 mM. d-Arabinose 5-phosphate is an effective inhibitor, while d-xylulose-5 phosphate is not, indicating that the configuration at carbon-3 contributes to substrate recognition. Although d-arabinose 5-phosphate binds to RPI, it is not isomerized, demonstrating that the configuration at carbon-2 is crucial for catalysis. Alignment of RPI sequences from diverse sources showed that only 11 charged amino acid residues of the 236-residue subunit are conserved. The possible function of four of these residues was examined by site-directed mutagenesis. D87A, K100A, and D90A mutants show greatly diminished kcat values (0.0012, 0.074, and 0.38% of the wild type, respectively), while E91A retains substantial activity. Only insignificant or moderate changes in Km of d-ribose 5-phosphate are observed for D87A, K100A, and D90A, indicating a direct or indirect catalytic role of the targeted residues.