We describe two families with retinitis pigmentosa (RP), a hereditary neurodegeneration of rod and cone photoreceptors in the retina, in which affected members were homozygotes for loss-of-function mutations in the IDH3B gene encoding the beta subunit of NAD-specific isocitrate dehydrogenase (NAD-IDH), the enzyme believed to catalyze the oxidation of isocitrate to α-ketoglutarate in the citric acid cycle. Cells from the affected family members had a substantial reduction of NAD-IDH activity with about a 300-fold increase in the Km for NAD. NADP-specific isocitrate dehydrogenase (NADP-IDH), an enzyme that can catalyze the same reaction, was normal. The patients had no health problems associated with the enzyme deficiency except for RP. The existence of these patients supports the hypothesis that mitochondrial NADP-specific IDH, rather than NAD-IDH, can serve as the major catalyst for this reaction in the citric acid cycle outside the retina, and that the retina has a particular requirement for NAD-IDH.Mutations in at least 34 genes have been identified as causes of nonsyndromic RP, including 15 dominant genes, 20 recessive genes, and 2 X-linked genes (RetNet website). The identified RP genes are estimated to account for about 60% of cases 1 . In addition, 11 unidentified genes have been mapped to specific chromosomal regions. It is estimated that there are dozens of still unmapped, unidentified recessive RP genes, each accounting for at most a few percent of cases 1, 2 .The approach we used to search for some of the unidentified recessive RP genes depends on the fact that recessive alleles are often deletions, frameshift mutations, or nonsense mutations that result in a scarcity of the disease gene's transcript either because the transcript is not made Correspondence to: Thaddeus P. Dryja, Massachusetts Eye and Ear Infirmary, 243 Charles Street, Boston, MA 02114; Thaddeus.dryja@novartis.com. * Current affiliation: University Medical Center Groningen, Groningen, The Netherlands § These authors contributed equally to the work AUTHOR CONTRIBUTIONS D.T.H., T.L.M., and T.P.D. designed and conducted the molecular genetic analyses. E.L.B. clinically evaluated, selected, and recruited patients and their family members, as well as helped in the design of the study. M.D. and R.F.C. designed and conducted the enzyme assays. All authors discussed and interpreted the results and wrote the manuscript. or is rapidly degraded due to nonsense-mediated decay 3 . The principle underlying this method has been previously described 4, 5, 6 , but, as far as we know, the method has never been used to identify a novel gene causing a hereditary disease. Using microarray techniques that simultaneously assay mRNA levels from tens of thousands of transcripts in individual patients, we searched for genes with absent or very low expression that may be due to two allelic diseasecausing mutations. While it would have been ideal to carry out the analysis with RNA derived from the retina prior to its degeneration, this tissue is not availa...
The human NAD-dependent isocitrate dehydrogenase (IDH) is a heterotetrameric mitochondrial enzyme with 2␣:1:1␥ subunit ratio. The three subunits share 40 -52% identity in amino acid sequence and each includes a tyrosine in a comparable position: ␣Y126, Y137, and ␥Y135. To study the role of the corresponding tyrosines of each of the subunits of human NAD-IDH, the tyrosines were mutated (one subunit at a time) to Ser, Phe, or Glu. Enzymes were expressed with one mutant and two wildtype subunits. The results of characterization of the mutant enzymes suggest that Y137 is involved in NAD binding and allosteric activation by ADP. The ␣Y126 is required for catalytic activity and likely acts as a general acid in the reaction. The ␥Y135 is also required for catalytic activity and may be involved in proper folding of the enzyme. The corresponding tyrosines in the three dissimilar subunits of NAD-IDH thus have distinctive functions. Mammalian NAD-dependent isocitrate dehydrogenase (NAD-IDH)2 is a mitochondrial tricarboxylic acid cycle enzyme that catalyzes the oxidative decarboxylation of isocitrate to ␣-ketoglutarate, while reducing NAD to NADH. It is a heterotetramer with three subunits in the ratio 2␣:1:1␥ (1), with molecular masses of ϳ37,000, 39,000, and 39,000 Da, respectively (2, 3). The amino acid sequences of the  and ␥ subunits of the human are 52.4% identical, whereas ␣ and  subunits are only 40.4% and ␣ and ␥ subunits are only 41.6% identical. This enzyme is allosterically regulated by ADP, which decreases the K m for isocitrate by ϳ38-fold (4). NAD-IDH has two binding sites per tetramer for each ligand: isocitrate, NAD, Mn 2ϩ , and ADP (2, 5), raising the question of the function of each of the subunits.We have recently shown that homozygous mutations exclusively of the  subunit of human NAD-IDH are a cause of Retinitis Pigmentosa, a hereditary degeneration of the retina that leads to blindness in patients (6). Characterization of these two types of mutant enzymes in lymphoblast cell extracts revealed a ϳ300-fold increase in K m,NAD and partial or complete loss of allosteric activation by ADP. The involvement of this enzyme in causing Retinitis Pigmentosa increases the importance of studying NAD-IDH in more detail.High-resolution crystal structures are available for the homodimeric NADP-dependent IDH of pig and Escherichia coli (7-11). The individual subunits of human NAD-IDH are only about 25-34% identical in amino acid sequence to the E. coli NADP-IDH (Fig. 1A) and about 12-18% identical to the pig NADP-IDH. Although there is a low % identity among these enzymes, the isocitrate-binding site is well conserved, including E. coli Arg-119, Arg-153, Tyr-160, and Lys-230. A previous study of pig NADP-IDH showed that Tyr-140 interacts with the -carboxylate of isocitrate and acts as a general acid in the reaction (8). The Tyr-160 mutants of E. coli NADP-IDH showed a decreased k cat (12, 13) and the crystal structure revealed that the tyrosine is appropriately positioned to donate a proton to the carbanion fo...
Mitochondrial NAD‐ and NADP‐isocitrate dehydrogenases (IDH) are crucial in the Krebs cycle. NAD‐IDH is composed of 3 subunits in the ratio 2α:1β:1γ and is allosterically regulated by ADP, by lowering the Km‐isocitrate. Two individuals with autosomal recessive Retinitis Pigmentosa (RP), but no other major symptoms, are homozygous for mutations in the β‐subunit of NAD‐IDH: the missense βL98P, and a 1‐basepair deletion in codon βI163 (ATT to –TT) resulting in a frameshift and premature termination of the β‐subunit. The activities of NAD‐ and NADP‐IDH were evaluated in extracts of lymphoblasts from unaffected individuals, heterozygote carriers of the mutations, and two RP patients. The NADP‐IDH activity was normal in all samples. In contrast, both homozygotes exhibited a Km‐NAD 260–300 times normal, while the Km‐NAD of the heterozygote samples was 4‐8 times that of normal. When ADP is added, Km‐isocitrate decreases only 3‐fold in the RP patient with the I163delA mutation, unlike normals in which ADP decreases Km‐isocitrate 9‐fold. This allosteric effect is completely lost in the homozygote βL98P mutant. The results show that these beta mutations primarily affect the enzyme's NAD affinity and ADP response. NADP‐IDH is the dominant mitochondrial IDH expressed in most tissues, while NAD‐IDH predominates in the eye. This differential expression may explain why the phenotype of NAD‐IDH mutations is confined to the eye.
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