Isolation and characterization of a cDNA encoding NADP(+)-specific isocitrate dehydrogenase from soybean (Glycine max).
A cDNA that encodes an NADP-specific isocitrate dehydrogenase (IDH) was cloned from a soybean nodule cDNA library by complementation of an Escherichia coli mutant that lacked IDH. DNA sequence analysis showed that the 1583 bp soybean cDNA could encode a protein that shares 63.9% amino acid sequence identity with the Saccharomyces cerevisiae NADP-IDH and long sequences of identity to an IDH from pig. Southern blot analysis suggests that this gene corresponds to a gene family made up of no more than two loci. The IDH cDNA hybridized to a 1.7 kb soybean mRNA and the relative amount of this transcript in soybean leaves, nodules and roots was 1:3.4:7.7. In alfalfa, a 1.7 kb mRNA was also found but the ratios for the corresponding tissues were 1:7.4:7.7. IDH activity was detected in the complemented E. coli strain and the electrophoretic mobility of this activity in nondenaturing polyacrylamide gels was identical to that of an IDH in extracts from soybean cotyledons or nodule cytosol. NADP-IDH specific activity in the E. coli host strain varied with growth phase; the highest rates (ca. 180 nmol/min per mg protein) were observed in late-stationary-phase cells. The enzyme had a broad pH optimum of 8.0 to 9.5 and had an absolute metal cofactor requirement, preferring Mn2+ below pH 8.0 and Mg2+ above pH 8.0. The Km for isocitrate and NADP was 21 microM and 11 microM respectively with Mn2+ as cofactor and 13 microM and 12 microM with Mg2+ as cofactor.
Udvardi
M K
MK
Institute of Biological Chemistry, Washington State University, Pullman 99164-6340.
McDermott
T R
TR
Kahn
M L
ML
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D004247:DNA / Q000302:isolation & purification; D004926:Escherichia coli; D015971:Gene Expression Regulation, Enzymologic; D007521:Isocitrate Dehydrogenase / Q000235:genetics* / Q000378:metabolism; D007700:Kinetics; D000455:Medicago sativa / Q000201:enzymology; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009586:Nitrogen Fixation / Q000235:genetics; D011994:Recombinant Proteins / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013025:Glycine max / Q000201:enzymology* / Q000235:genetics; D013347:Subcellular Fractions / Q000201:enzymology
D011994:Recombinant Proteins; D004247:DNA; D007521:Isocitrate Dehydrogenase; C039355:isocitrate dehydrogenase (NADP+)