Characterization of native and histidine-tagged deoxyxylulose 5-phosphate reductoisomerase from the cyanobacterium Synechocystis sp. PCC6803.
The dxr gene encoding the 1-deoxy-D-xylulose 5-phosphate reductoisomerase (DXR) from the cyanobacterium Synechocystis sp. PCC6803 was expressed in Escherichia coli to produce both the native and N-terminal histidine-tagged forms of DXR. The enzymes were purified from the cell extracts using either anion exchange chromatography or metal affinity chromatography and gel filtration. The purified recombinant native and histidine-tagged enzymes each displayed a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels, corresponding to the calculated subunit molecular weights of 42,500 and 46,700, respectively. By native PAGE, both enzymes were dimers under reducing conditions. The kinetic properties for the enzymes were characterized and only minor variations were observed, demonstrating that the N-terminal histidine tag does not greatly affect the activity of the enzyme. Both enzymes had similar properties to previously characterized reductoisomerases from other sources. The K(m)'s for the metal ions Mn(2+), Mg(2+), and Co(2+) were determined for native DXR for the first time, with the K(m) for Mg(2+) being approximately 200-fold higher than the K(m)'s for Mn(2+) and Co(2+).
College of Pharmacy, Pharmacy Building, Room 203, Oregon State University, Corvallis, OR 97331-3507, USA.
Xihou
X
Yin
Philip J
PJ
Proteau
D019747:Aldose-Ketose Isomerases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D003035:Cobalt / Q000737:chemistry / Q000378:metabolism; D000458:Cyanobacteria / Q000201:enzymology* / Q000235:genetics; D019281:Dimerization; D004926:Escherichia coli / Q000378:metabolism; D006639:Histidine / Q000737:chemistry*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008274:Magnesium / Q000737:chemistry / Q000378:metabolism; D008345:Manganese / Q000737:chemistry / Q000378:metabolism; D009097:Multienzyme Complexes / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D010088:Oxidoreductases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D021122:Protein Subunits; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D013302:Streptomyces / Q000201:enzymology; D013696:Temperature; D017355:Zymomonas / Q000201:enzymology
D009097:Multienzyme Complexes; D021122:Protein Subunits; D011994:Recombinant Proteins; D003035:Cobalt; D008345:Manganese; D006639:Histidine; D010088:Oxidoreductases; C114231:1-deoxy-D-xylulose 5-phosphate reductoisomerase; D019747:Aldose-Ketose Isomerases; D008274:Magnesium