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 K(i) 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 k(cat) values (0. 0012, 0.074, and 0.38% of the wild type, respectively), while E91A retains substantial activity. Only insignificant or moderate changes in K(m) of D-ribose 5-phosphate are observed for D87A, K100A, and D90A, indicating a direct or indirect catalytic role of the targeted residues.
Jung
C H
CH
Life Sciences Division, The University of Tennessee-Oak Ridge Graduate School of Biomedical Sciences, Oak Ridge, Tennessee 37831-2009, USA.
Hartman
F C
FC
Lu
T Y
TY
Larimer
F W
FW
D019747:Aldose-Ketose Isomerases / Q000737:chemistry / Q000235:genetics*; D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D019281:Dimerization; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004926:Escherichia coli; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010710:Phosphates / Q000494:pharmacology; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics; D016415:Sequence Alignment; D018724:Spinacia oleracea / Q000201:enzymology*
D004791:Enzyme Inhibitors; D010710:Phosphates; D011994:Recombinant Proteins; D019747:Aldose-Ketose Isomerases; C022548:ribosephosphate isomerase