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) wa s cloned and overexpressed in Escherichia coli, and a purification scheme f or 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 K-i of 7.9 mM, D-Arabinose 5-phos phate is an effective inhibitor, while D-xylulose-5 phosphate is not, indic ating that the configuration at carbon-3 contributes to substrate recogniti on, Although D-arabinose 5-phosphate binds to RPI, it is not isomerized, de monstrating that the configuration at carbon-3 is crucial for catalysis. Al ignment of RPI sequences from diverse sources showed that only 11 charged a mino acid residues of the 236-residue subunit are conserved. The possible f unction of four of these residues was examined by site-directed mutagenesis . D87A, K100A, and D90A mutants show greatly diminished k(cat) values (0.00 12, 0.074, and 0.38% of the wild type, respectively), while E91A retains su bstantial activity. Only insignificant or moderate changes in K-m of D-ribo se 5-phosphate are observed for D87A, K100A, and D90A, indicating a direct or indirect catalytic role of the targeted residues. (C) 2000 Academic Pres s.