CHARACTERIZATION OF A CDNA CLONE FOR HUMAN NAD(-SPECIFIC ISOCITRATE DEHYDROGENASE ALPHA-SUBUNIT AND STRUCTURAL COMPARISON WITH ITS ISOENZYMES FROM DIFFERENT SPECIES())
Yo. Kim et al., CHARACTERIZATION OF A CDNA CLONE FOR HUMAN NAD(-SPECIFIC ISOCITRATE DEHYDROGENASE ALPHA-SUBUNIT AND STRUCTURAL COMPARISON WITH ITS ISOENZYMES FROM DIFFERENT SPECIES()), Biochemical journal, 308, 1995, pp. 63-68
A 0.6 kb cDNA fragment encoding the human NAD(+)-specific isocitrate d
ehydrogenase alpha-subunit (H-IDH alpha) was amplified by PCR using ol
igonucleotide primers synthesized on the basis of pig tryptic peptide
sequences [Huang and Colman (1990) Biochemistry 29, 8266-8273]. With t
he amplified cDNA as a probe, cDNA clones for IDH alpha were isolated
from a human heart lambda gt11 cDNA library, The deduced protein seque
nce of the largest cDNA clone (2628 bp) rendered a precursor protein o
f 366 amino acids (39 591 Dal and a mature protein of 339 amino acids
(36640 Dal. The deduced H-IDH alpha protein sequence is highly similar
to the partial peptide sequences of the pig enzyme, It is 55, 43 and
44% identical with yeast NAD(+)-specific IDH2, yeast NAD(+)-specific I
DH1 and monkey NAD(+)-specific IDH gamma-subunit (IDH gamma) respectiv
ely. However, it has less similarity (about 3.0%) to NADP(+)-specific
IDH from Escherichia coli and bovine mitochondria. These results indic
ate that the structure of IDH alpha closely resembles that of IDH2, th
e catalytic subunit of the yeast enzyme, Structural analysis of the de
duced H-IDH alpha protein revealed that the amino acids responsible fo
r the binding of isocitrate, Mg2+ and NAD are highly conserved, It als
o has two conserved motifs for the binding sites of ATP and ADP, but a
canonical Ca2+-binding motif was not recognized. Unusual penta- (ATTT
A) and tri-(TAA or ATT) nucleotides which are respectively believed to
interact with RNA-binding proteins and be near the endonuclease cleav
age sites were frequently recognized in its 3' untranslated region, in
dicating the possibility of an additional method of regulation of this
enzyme. Northern-blot analysis suggests that one mRNA transcript (2.8
kb) exists in cultured HeLa cells. Genomic DNA Southern-blot analysis
indicates that the IDH alpha gene is not closely related to that of t
he other IDH isoenzymes, and IDH alpha appears to be encoded by a sing
le gene.