Kyoko Nakano

The α-ketoacid dehydrogenase complexes. Sequence similarity of rat pyruvate dehydrogenase with Escherichia coli and Azotobacter vinelandii α-ketoglutarate dehydrogenase

Abstract The pyruvate dehydrogenase complex and the α-ketoglutarate dehydrogenase complex are multienzyme complexes consisting of three different enzymes. No significant similarity has been reported among the dehydrogenases which are component enzymes of these complexes, despite the presence of homology among the other component enzymes. Here we isolated cDNAs for the α and β subunits of rat pyruvate dehydrogenase and they exhibited a significant similarity of the amino acid sequences among rat pyruvate dehydrogenase, 2-oxoisovalerate dehydrogenase (which is a dehydrogenase component of branched chain α-ketoacid dehydrogenase complex) and α-ketoglutarate dehydrogenase, suggesting that they have been derived from a common ancestral dehydrogenase. Our results suggested that the α and β subunits of the pyruvate and 2-oxoisovalerate dehydrogenases have been derived by the cleavage of the α-ketoglutarate dehydrogenase. However, we could not find significant homology between rat pyruvate dehydrogenase and Gram-negative bacterial pyruvate dehydrogenase.

Molecular cloning of dihydrolipoamide acetyltransferase of the rat pyruvate dehydrogenase complex: sequence comparison and evolutionary relationship to other dihydrolipoamide acyltransferases

A complementary DNA (cDNA) clone of dihydrolipoamide acetyltransferase (E2) of the rat pyruvate dehydrogenase complex (PDC) was isolated from a lambda gt11 rat heart cDNA library. The amino acid sequence of a full mature protein of rat PDC-E2 was predicted by combination of the cDNA nucleotide sequence and the N-terminal amino acid sequence determined chemically. The amino acid sequence of rat PDC-E2 was well consistent with those of the E2 components of other alpha-ketoacid dehydrogenase complexes. These E2 components possess the sequence G-X-G-X-X-G, which is the consensus sequence for nucleotide binding sites of nucleotide binding proteins, in the E3 and/or E1 binding domains. The E2 components of the three alpha-ketoacid dehydrogenase complexes are suggested to be classified into three clusters separated during evolution.

Human dihydrolipoamide succinyltransferase: cDNA cloning and localization on chromosome 14q24.2-q24.3

We isolated cDNA for dihydrolipoamide succinyltransferase from a human fibroblast cDNA library in lambda gt11. The cDNA revealed that the human dihydrolipoamide succinyltransferase lacked a sequence motif of an E3 and/or E1 binding site. This suggests that the human dihydrolipoamide succinyltransferase possesses a unique structure consisting of two domains in contrast with the dihydrolipoamide acyltransferases of other alpha-keto acid dehydrogenase complexes. In addition, we found that the human dihydrolipoamide succinyltransferase gene is located on chromosome 14 at q24.2-q24.3 and that a sequence related to the dihydrolipoamide succinyltransferase gene is located on chromosome 1 at p31. Interestingly, the gene for the dihydrolipoamide acyltransferase of the branched chain alpha-keto acid dehydrogenase complex is also located on chromosome 1p31 (Zneimer et al. (1991) Genomics 10, 740-747).

Truncated product of the bifunctional DLST gene involved in biogenesis of the respiratory chain.

Dihydrolipoamide succinyltransferase (DLST) is a subunit enzyme of the α‐ketoglutarate dehydrogenase complex of the Krebs cycle. While studying how the DLST genotype contributes to the pathogenesis of Alzheimers disease (AD), we found a novel mRNA that is transcribed starting from intron 7 in the DLST gene. The novel mRNA level in the brain of AD patients was significantly lower than that of controls. The truncated gene product (designated MIRTD) localized to the intermembrane space of mitochondria. To investigate the function of MIRTD, we established human neuroblastoma SH‐SY5Y cells expressing a maxizyme, a kind of ribozyme, that specifically digests the MIRTD mRNA. The expression of the maxizyme specifically eliminated the MIRTD protein and the resultant MIRTD‐deficient cells exhibited a marked decrease in the amounts of subunits of complexes I and IV of the mitochondrial respiratory chain, resulting in a decline of activity. A pulse‐label experiment revealed that the loss of the subunits is a post‐translational event. Thus, the DLST gene is bifunctional and MIRTD transcribed from the gene contributes to the biogenesis of the mitochondrial respiratory complexes.

Molecular cloning of cDNA for rat liver lipoate acetyltransferase. A component of pyruvate dehydrogenase complex.

One cDNA clone for lipoate acetyltransferase, a component enzyme of pyruvate dehydrogenase complex, was isolated from a rat liver cDNA library prepared in the phage expression vector lambda gt11 using immunological screening with affinity purified anti-lipoate acetyltransferase antibody. It was identified tha cDNA insert in this clone codes for lipoate acetyltransferase by immunoblotting of lysogen carrying the isolated clone. Lipoate acetyltransferase antigenic polypeptide in fusion protein was about 11,000 daltons, agreeing with the size of cDNA insert to be 300 base pairs.

Isolation and sequence analysis of the rat dihydrolipoamide succinyltransferase gene.

The dihydrolipoamide succinyltransferase (DLST) gene of the f -ketoglutarate dehydrogenase complex ( f -KGDC) was isolated from a rat genomic DNA library and sequenced. This gene was composed of 15 exons and 14 introns like the human DLST gene. Sequence analysis of the promoter-regulatory region of the rat DLST gene- ( Dlst ) showed the possible presence of a CAAT box-sequence and of the sequences for an AP-2 site and three Sp1 sites, but no TATA box-sequence was evidenced. The nucleotide sequences of introns 1 and 4 of the rat Dlst were significantly homologous to those of introns 1 and 4 of the human DLST gene. The sequence analysis of the rat Dlst suggested that the exon coding for the E3- and/or E1-binding domain may have been lost from the gene during evolution in eukaryotic DLST, possibly after mitochondrial symbiosis because prokaryotic DLST possesses the E3- and/or E1-binding domain.

A novel protein found in the I bands of myofibrils is produced by alternative splicing of the DLST gene

BACKGROUND It is not known if the dihydrolipoamide succinyltransferase (DLST) gene, a mitochondrial protein, undergoes alternative splicing. We identified an uncharacterized protein reacting with an anti-DLST antibody in the I bands of myofibrils in rat skeletal muscle. METHODS Immunocytochemical staining with an anti-DLST antibody, the purification and amino acid sequence analysis of the protein, and the isolation and sequencing of the proteins cDNA were carried out to clarify the properties of the protein and its relationship to the DLST gene. RESULTS A pyrophosphate concentration >10 mM was necessary to extract the protein from myofibrils in the presence of salt with a higher concentration than 0.6 M, at an alkaline pH of 7.5-8.0. The protein corresponded to the amino acid sequence of the C-terminal side of DLST. The cDNAs for this protein were splicing variants of the DLST gene, with deletions of both exons 2 and 3, or only exon 2 or 3. These variants possessed an open reading frame from an initiation codon in exon 8 of the DLST gene to a termination codon in exon 15, generating a protein with a molecular weight of 30 kDa. CONCLUSIONS The DLST gene undergoes alternative splicing, generating the protein isolated from the I bands of myofibrils. GENERAL SIGNIFICANCE The DLST gene produces two different proteins with quite different functions via alternative splicing.

E2-cDNA cloning and component X of pyruvate dehydrogenase complex

Pyruvate dehydrogenase complex (PDH complex) consists of three component enzymes, pyruvate dehydrogenase (E~; EC 1.2.4.1), lipoate acetyltransferase (E2; EC 2.3.1.12) and lipoamide dehydrogenase (g3; EC 1.6.4.3) (Linnet al., 1972). Many cases of PDH complex deficiency have been reported. These are mainly due to E~ deficiency (Ho et al., 1986) but deficiency of E2 has been reported in one case (Cederbaum et al., 1976). Recently, many reports suggest the presence of a new component (which is referred to as component X or protein X) in the mammalian PDH complex in addition to the three component enzymes (DeMarcucci and Lindsay, 1985; DeMarcucci et al., 1985; Jilka et al., t986; Rahmatullah and Roche, 1987). As to the physiological role of component X, Rahmatullah and Roche (1987) have reported that it not only serves to activate kinase activity with its acetylation, but also serves to anchor the kinase to the core of the complex. However, there is no direct evidence that component X is not a proteolytic-degradative product of E 2. It should be determined whether component X is coded for by its own mRNA. Now we have isolated cDNA of 1700 base pairs for E2. Here we report the properties of component X in rat PDH complex and the cloning of Ez-cDNA from rat.

Properties of component X of rat heart pyruvate dehydrogenase complex

Pyruvate dehydrogenase complex was purified from rat heart. A new component(mol.wt; 52,000) was found in the purified complex in addition to well known three component enzymes. This component(referred to as component X) was acetylated with [2-14C] pyruvate in the absence of CoA as well as lipoate acetyltransferase. The anti-lipoate acetyltransferase antibody reacted with component X and lipoate acetyltransferase, suggesting that component X shows homology with lipoate acetyltransferase in protein structure. cDNA for lipoate acetyltransferase was isolated from rat liver cDNA library in lambda gt 11. cDNA for lipoate acetyltransferase recognized two kinds of mRNAs of 3.5 Kb and 2.5 Kb.