Isolation and characterization of a cDNA encoding NADP+-specific isocitrate dehydrogenase from soybean (Glycine max)

Abstract

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.

Cloning and Expression Analysis of the Cytosolic NADP+-Dependent Isocitrate Dehydrogenase from Potato (Implications for Nitrogen Metabolism)

“…ICDH is encoded by either one gene or a small gene family (see also Shorrosh and Dixon, 1992;Udvardi et al, 1993). A hydropathy plot (Kyte and Doolittle, 1982) of the inferred potato protein revealed a predominance of hydrophilic amino acids (data not shown), making a tight membrane association of the enzyme unlikely.…”

“…The overall sequence identity amounts to about 97% for tobacco (S. Gálvez, EMBL Data Library, accession No. X77944), to 90% for soybean (Udvardi et al, 1993) and alfalfa (Shorrosh and Dixon, 1992), to more than 65% for cattle (Huh et al, 1993), pig (Haselbeck et al, 1992), and yeast (Haselbeck and McAlister-Henn, 1991), and finally to 25% for Escherickiu coli (Thorsness and Koshland, 1987). Southern blot analysis of digested genomic DNA from potato probed with the complete icdh-2 cDNA fragment showed only a few bands (Fig.…”

“…Only recently has plant ICDH been analyzed at the DNA and RNA levels (Shorrosh and Dixon, 1992;Udvardi et al, 1993), opening new possibilities for a deeper understanding of the role of this enzyme during plant growth and development. Here, we report the isolation of a cDNA clone encoding a cytosolic NADP+-dependent ICDH from potato (Solanum tuberosum L.).…”

Cloning, sequence analysis, expression, and inactivation of the Corynebacterium glutamicum icd gene encoding isocitrate dehydrogenase and biochemical characterization of the enzyme

“…strain PCC7120 (35), and Bacillus subtilis (26), as well as from some eucaryotes (7,17,18,21,54). Comparison of the deduced amino acid sequences revealed conserved regions in all ICDs (7,35).…”

Purification and Characterization of NAD-Isocitrate Dehydrogenase from Chlamydomonas reinhardtii1

“…Cytosolic NADP-IDH has been purified from higher plants (Chen et al, 1988) and eukaryotic algae (Martínez-Rivas et al, 1996), and its cDNA has been cloned from alfalfa (Shorrosh and Dixon, 1992), soybean (Udvardi et al, 1993), potato (Fieuw et al, 1995), and tobacco (Gálvez et al, 1996). This 80-kD isoenzyme is a dimer, and it is likely to be involved in the synthesis of NADPH for biosynthetic purposes in the cytosol (Chen et al, 1988), in the synthesis of 2-oxoglutarate for ammonium assimilation (Chen and Gadal, 1990b), and in the cycling, redistribution, and export of amino acids (Fieuw et al, 1995).…”