Molecular cloning and deduced amino acid sequences of the <i>α</i>- and <i>β</i>- subunits of mammalian NAD+-isocitrate dehydrogenase

Nichols, Benjamin J.; Perry, Anthony C.F.; Hall, Len; Denton, Richard M.

doi:10.1042/bj3100917

Cited by 37 publications

(42 citation statements)

References 31 publications

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“…The deduced amino acid sequence of the protein encoded by S. mutans icd was most similar (72%) to the ICDH from the closely related oral organism Streptococcus salivarius that has been reported to be an NAD ϩ -dependent enzyme (10). There is very little homology (32%) with sequences recently obtained (30) for the NAD ϩ -dependent ICDH from the primate Macaca fascicularis. A search of the genetic database revealed no greater homology than 32% when comparing the S. mutans ICDH with analogous eukaryotic enzymes.…”

Section: Resultsmentioning

confidence: 99%

Role of the citrate pathway in glutamate biosynthesis by Streptococcus mutans

1997

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. 178:4166-4175, 1996), a Tn917 transposon-generated mutant of Streptococcus mutans JH1005 unable to synthesize glutamate anaerobically was isolated and the insertion point of the transposon was determined to be in the icd gene encoding isocitrate dehydrogenase (ICDH). The intact icd gene of S. mutans has now been isolated from an S. mutans genomic plasmid library by complementation of an icd mutation in Escherichia coli host strain EB106. Genetic analysis of the complementing plasmid pJG400 revealed an open reading frame (ORF) of 1,182 nucleotides which encoded an enzyme of 393 amino acids with a predicted molecular mass of 43 kDa. The nucleotide sequence contained regions of high (60 to 72%) homology with icd genes from three other bacterial species. Immediately 5 of the icd gene, we discovered an ORF of 1,119 nucleotides in length, designated citZ, encoding a homolog of known citrate synthase genes from other bacteria. This ORF encoded a predicted protein of 372 amino acids with a molecular mass of 43 kDa. Furthermore, plasmid pJG400 was also able to complement a citrate synthase (gltA) mutation of E. coli W620. The enzyme activities of both ICDH, found to be NAD ؉ dependent, and citrate synthase were measured in cell extracts of wild-type S. mutans and E. coli mutants harboring plasmid pJG400. The region 5 from the citZ gene also revealed a partial ORF encoding 264 carboxy-terminal amino acids of a putative aconitase gene. The genetic and biochemical evidence indicates that S. mutans possesses the enzymes required to convert acetyl coenzyme A and oxalacetate to ␣-ketoglutarate, which is necessary for the synthesis of glutamic acid. Indeed, S. mutans JH1005 was shown to assimilate ammonia as a sole source of nitrogen in minimal medium devoid of organic nitrogen sources.

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Section: Resultsmentioning

confidence: 99%

Role of the citrate pathway in glutamate biosynthesis by Streptococcus mutans

1997

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“…IPMDH contains two cysteine residues, Cys167 and Cys185. Because one of them, Cys185, is conserved in several mitochondrial NAD-dependent ICDHs (all subunits of the monkey enzyme [26], 3/4 subunit of the bovine enzyme [41], and ␥ subunits of the pig [11] and the rat [27] enzymes), it is possible that these Cys residues form an intersubunit disulfide bond, contributing to the formation of their quaternary structure. To investigate this possibility, a quantitative analysis of free SH groups was performed.…”

Section: Resultsmentioning

confidence: 99%

Molecular and phylogenetic characterization of isopropylmalate dehydrogenase of a thermoacidophilic archaeon, Sulfolobus sp. strain 7

et al. 1997

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The archaeal leuB gene encoding isopropylmalate dehydrogenase of Sulfolobus sp. strain 7 was cloned, sequenced, and expressed in Escherichia coli. The recombinant Sulfolobus sp. enzyme was extremely stable to heat. The substrate and coenzyme specificities of the archaeal enzyme resembled those of the bacterial counterparts. Sedimentation equilibrium analysis supported an earlier proposal that the archaeal enzyme is homotetrameric, although the corresponding enzymes studied so far have been reported to be dimeric. Phylogenetic analyses suggested that the archaeal enzyme is homologous to mitochondrial NAD-dependent isocitrate dehydrogenases (which are tetrameric or octameric) as well as to isopropylmalate dehydrogenases from other sources. These results suggested that the present enzyme is the most primitive among isopropylmalate dehydrogenases belonging in the decarboxylating dehydrogenase family.3-Isopropylmalate dehydrogenase (IPMDH; EC 1.1.1.85) is the third enzyme in the leucine biosynthetic pathway and catalyzes the oxidative decarboxylation of threo-D S -3-isopropylmalate to 2-oxoisocaproate (32). Genes coding for the enzyme have been cloned and sequenced from various bacteria (eubacteria) and eukarya (eukaryotes). IPMDHs so far investigated are NAD dependent and homodimeric (20,39).It has been suggested that the catalytic mechanism of IPMDH is similar to that of bacterial homodimeric NADPdependent isocitrate dehydrogenase (ICDH; EC 1.1.1.42) a key enzyme in the tricarboxylic acid cycle. The latter catalyzes the oxidative decarboxylation of threo-D S -isocitrate to oxoglutarate (9). The primary structures of IPMDHs are homologous to those of the homodimeric bacterial NADP-dependent ICDHs (42). In addition, IPMDHs show sequence similarities to mitochondrial NAD-dependent ICDHs. The eukaryotic NAD-dependent ICDHs are either heterotetrameric (e.g., ␣2␤␥ for the mammalian mitochondrial enzymes) (33, 41) or heterooctameric (e.g., ␣4␤4 for the yeast mitochondrial enzyme) (21), consisting of subunits homologous to each other.Recently, the X-ray crystal structure of Thermus thermophilus HB8 IPMDH has been determined (13,15,19). The structure resembles that of Escherichia coli 14). In particular, both enzymes are unique in lacking the Rossmann fold motif which is frequently found in NAD(P)-dependent enzymes. In addition, most of the functional key residues proposed by the crystallographic studies are conserved among IPMDHs, bacterial NADP-dependent ICDHs, and mitochondrial NAD-dependent ICDHs (42). The structural evidence strongly supports the idea (13, 42) that these enzymes originated from a gene duplication that preceded the divergence of the three domains of life.To clarify the phylogenetic relationship among IPMDHs and ICDHs, the structural and functional data of archaeal enzyme are absolutely required, since Archaea (archaebacteria) comprises the third independent domain of life, which is only distantly related to both the Bacteria and the Eukarya (38). Recently, we have isolated and preliminarily characterize...

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“…However, these amino acids are not involved in the active site for NAD ϩ , because this substrate did not protect against inactivation. Arg and Lys residues are conserved in the deduced amino acid sequences of the corresponding NAD-IDH cDNA clones from yeast (Cupp andMcAllister-Henn, 1991, 1992) and animals (Nichols et al, 1995;Zeng et al, 1995). The presence of these residues in the active site for the isocitrate-divalent cation substrate, but not for NADP ϩ , has been well established for the NADP-IDH from Escherichia coli (Hurley et al, 1989).…”

Section: Figurementioning

confidence: 99%

“…The NAD-IDH from yeast is activated by AMP and citrate (Hathaway and Atkinson, 1963), whereas the animal enzyme is activated by ADP and citrate (Cohen and Colman, 1972). In addition, the NAD-IDH cDNAs have been cloned from yeast (Cupp andMcAlister-Henn, 1991, 1992) and animals (Nichols et al, 1995;Zeng et al, 1995). In these organisms, the enzyme is composed of two (yeast) or more (animals) different subunits encoded by different genes.…”

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confidence: 99%

Purification and Characterization of NAD-Isocitrate Dehydrogenase from Chlamydomonas reinhardtii1

Martínez-Rivas¹,

Vega

1998

Plant Physiology

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NAD-isocitrate dehydrogenase (NAD-IDH) from the eukaryotic microalga Chlamydomonas reinhardtii was purified to electrophoretic homogeneity by successive chromatography steps on Phenyl-Sepharose, Blue-Sepharose, diethylaminoethyl-Sephacel, and Sephacryl S-300 (all Pharmacia Biotech). The 320-kD enzyme was found to be an octamer composed of 45-kD subunits. The presence of isocitrate plus Mn 2؉ protected the enzyme against thermal inactivation or inhibition by specific reagents for arginine or lysine. NADH was a competitive inhibitor (K i , 0.14 mM) and NADPH was a noncompetitive inhibitor (K i , 0.42 mM) with respect to NAD ؉ . Citrate and adenine nucleotides at concentrations less than 1 mM had no effect on the activity, but 10 mM citrate, ATP, or ADP had an inhibitory effect. In addition, NAD-IDH was inhibited by inorganic monovalent anions, but L-amino acids and intermediates of glycolysis and the tricarboxylic acid cycle had no significant effect. These data support the idea that NAD-IDH from photosynthetic organisms may be a key regulatory enzyme within the tricarboxylic acid cycle.

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Molecular cloning and deduced amino acid sequences of the α- and β- subunits of mammalian NAD+-isocitrate dehydrogenase

Cited by 37 publications

References 31 publications

Role of the citrate pathway in glutamate biosynthesis by Streptococcus mutans

Role of the citrate pathway in glutamate biosynthesis by Streptococcus mutans

Molecular and phylogenetic characterization of isopropylmalate dehydrogenase of a thermoacidophilic archaeon, Sulfolobus sp. strain 7

Purification and Characterization of NAD-Isocitrate Dehydrogenase from Chlamydomonas reinhardtii1

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