Purification, properties and enhanced expression under nitrogen starvation of the NADP+-isocitrate dehydrogenase from the cyanobacterium Phormidium laminosum.
Nitrogen starvation enhances up to 8-fold the cellular level of the NADP+-dependent isocitrate dehydrogenase activity (isocitrate:NADP+ oxidoreductase (decarboxylating), IDH, EC 1.1.1.42) in the thermophilic filamentous non-N2-fixing cyanobacterium Phormidium laminosum. The enzyme was purified 650-fold to electrophoretic homogeneity from nitrogen-starved cells with an activity yield of 25% and a specific activity of 500 U (mg protein)-1. The native enzyme showed a pI of 5.9 and it was a dimer of 107 kDa consisting of two identical subunits of 53 kDa. The activity required the presence of a divalent metal cation as an essential activator, Mn2+ or Mg2+ being the most effective. The optimum temperature for activity was 55 degrees C and the Ea for catalysis was 39.7 kJ mol-1. An optimum pH for activity of 8.5 was found and the calculated pKE1, pKE2 and pKES1 of enzyme ionisation groups were 6.0, 8.9 and 6.3, respectively. Km values of 22, 50 and 24 microM were calculated for d,l-isocitrate, NADP and Mn2+, respectively, in the Mn2+-dependent reaction and 70, 32 and 159 microM for d,l-isocitrate, NADP and Mg2+, respectively, in the Mg2+-dependent reaction. The decarboxylating activity was inhibited by ATP, ADP and by its reaction products 2-oxoglutarate and NADPH2. Polyclonal antibodies raised against the pure IDH were used to assess the presence of the enzyme in cells subjected to nitrogen starvation.
Pardo
M A
MA
Enzyme and Cell Technology Group, Department of Biochemistry and Molecular Biology, University of the Basque Country, P.O. Box 644, E-48080, Bilbao, Spain.
Llama
M J
MJ
Serra
J L
JL
D000458:Cyanobacteria / Q000201:enzymology*; D006863:Hydrogen-Ion Concentration; D007521:Isocitrate Dehydrogenase / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification*; D007526:Isoelectric Point; D008970:Molecular Weight; D009566:Nitrates; D009584:Nitrogen; D013696:Temperature
D009566:Nitrates; D007521:Isocitrate Dehydrogenase; C039355:isocitrate dehydrogenase (NADP+); D009584:Nitrogen