Kinetic characterization of Synechocystis sp. PCC6803 1-deoxy-D-xylulose 5-phosphate reductoisomerase mutants.
The methylerythritol phosphate pathway to isoprenoids has been firmly established as an alternate to the mevalonate pathway in many bacteria, plants, algae, and the malaria parasite Plasmodium falciparum. The second enzyme in this pathway, deoxy-D-xylulose 5-phosphate reductoisomerase (DXR; E.C. 1.1.1.267), has been the focus of many investigations since it was found to be the target of the antibacterial and antimalarial compound, fosmidomycin. Several x-ray crystal structures of the Escherichia coli and Zymomonas mobilis DXR enzymes have provided important structural information about the residues potentially involved in substrate binding and catalysis. Site-directed mutagenesis studies can be used to complement the structural studies, providing kinetic data for specific changes of active site residues. Active site mutants were prepared of the recombinant Synechocystis sp. PCC6803 DXR, targeting residues D152, S153, E154, H155, M206, and E223. Alteration of the three acidic residues had major effects on catalysis, changes to S153 and M206 had variable effects on binding and catalysis, and a H155A mutation had only minimal effects on the kinetic parameters.
Fernandes
Roberta P M
RP
Department of Pharmaceutical Sciences, College of Pharmacy, Pharmacy Bldg. Rm. 203, Oregon State University, Corvallis, OR 97331-3507, USA.
Proteau
Philip J
PJ
D019747:Aldose-Ketose Isomerases / Q000737:chemistry* / Q000235:genetics*; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001665:Binding Sites; D007700:Kinetics; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000737:chemistry* / Q000235:genetics*; D009154:Mutation; D010088:Oxidoreductases / Q000737:chemistry* / Q000235:genetics*; D011487:Protein Conformation; D046939:Synechocystis / Q000201:enzymology*
D009097:Multienzyme Complexes; D010088:Oxidoreductases; C114231:1-deoxy-D-xylulose 5-phosphate reductoisomerase; D019747:Aldose-Ketose Isomerases