Molecular characterization of higher plant NAD-dependent isocitrate dehydrogenase: evidence for a heteromeric structure by the complementation of yeast mutants

Citation
M. Lancien et al., Molecular characterization of higher plant NAD-dependent isocitrate dehydrogenase: evidence for a heteromeric structure by the complementation of yeast mutants, PLANT J, 16(3), 1998, pp. 325-333
Citations number
47
Language
INGLESE
art.tipo
Article
Categorie Soggetti
Plant Sciences","Animal & Plant Sciences
Journal title
PLANT JOURNAL
ISSN journal
0960-7412 → ACNP
Volume
16
Issue
3
Year of publication
1998
Pages
325 - 333
Database
ISI
SICI code
0960-7412(199811)16:3<325:MCOHPN>2.0.ZU;2-H
Abstract
NAD-dependent isocitrate dehydrogenase (IDH) is a key enzyme controlling th e activity of the citric acid cycle. Despite more than 30 years of work, th e plant enzyme remains poorly characterized. In this paper, a molecular cha racterization of the plant IDH is presented. Starting from probes defined a ccording to sequence comparisons, three full-length cDNAs named Ntidha Ntid hb and Ntidhc encoding different IDH subunits have been isolated from a Nic otiana tabacum cell suspension library. Sequence comparisons of the tobacco IDH subunits with the E. coli NADP-dependent enzyme, and the yeast IDH1 an d IDH2 subunits suggested that only IDHa had the capacity to be catalytic a s IDHb and IDHc were lacking certain residues implied in catalysis. The abi lity of antibodies raised against the recombinant IDHa protein to preferent ially cross-react with IDH2 indicated that IDHa was more closely related to IDH2 than to IDH1. Complementation of yeast single IDH mutants showed that IDHb and IDHc could replace the function of the yeast regulatory IDH1 subu nit. Although IDHa was unable to complement the IDH2 mutant, its catalytic function was revealed by the ability of two heteromeric enzymes, composed o f either IDHa with IDHb or IDHa with IDHc, to replace IDH function in a yea st double mutant lacking both subunits. Expression studies at the protein a nd mRNA levels show that each subunit is present in both root and leaf tiss ues and that the three IDH genes respond in the same way to nitrate additio n. Taken together, such observations suggest that the physiologically activ e enzyme is composed of the three different subunits. These results show fo r the first time that the plant IDH is heteromeric and that IDH subunit com position appears to be conserved between plant and animal kingdoms.