The variant human isovaleryl-CoA dehydrogenase gene responsible for type II isovaleric acidemia determines an RNA splicing error, leading to the deletion of the entire second coding exon and the production of a truncated precursor protein that interacts poorly with mitochondrial import receptors.

Isovaleryl-CoA dehydrogenase (IVD) is a mitochondrial enzyme involved in leucine metabolism. Previous studies of fibroblasts from patients with isovaleric acidemia (IVA), an inherited defect in IVD, have revealed that IVD precursor protein produced by type II IVA cells is 3 kDa smaller than normal and is processed inefficiently to a mature form which is also 3 kDa smaller than normal. Using the polymerase chain reaction, we have identified a 90-base pair deletion encompassing bases 145-234 in type II IVD cDNA. This deletion is caused by an error in RNA splicing and predicts the in-frame deletion of 30 amino acids beginning with leucine 20 of the mature IVD. The rate of leader peptide cleavage by purified mitochondrial leader peptidases was similar for the variant and normal precursor IVDs expressed in vitro, and radiosequencing confirmed that both mature proteins contain identical amino termini. In vitro import studies showed that the efficiency of overall mitochondrial import of type II variant IVD precursor was approximately 30% of normal, as was its binding to the mitochondrial surface. Unlike its normal counterpart, the bound variant IVD precursor was readily released. These data suggest that binding of the variant protein to mitochondrial membrane receptors per se is hindered, resulting in the inefficient mitochondrial processing.     

Molecular basis of isovaleric acidemia and medium-chain acyl-CoA dehydrogenase deficiency

Our early study of isovaleric acidemia (IVA) indicated that isovaleryl-CoA is dehydrogenated by an enzyme that is specific for isovaleryl-CoA. We subsequently identified and purified isovaleryl-CoA dehydrogenase (IVD) and 2-methyl-branched chain acyl-CoA dehydrogenase, which were previously unknown. We also purified and characterized three previously known acyl-CoA dehydrogenases. Five acyl-CoA dehydrogenases share similar molecular features and reaction mechanisms, indicating a close evolutionary relationship. Using the tritium release assay and [35S]methionine labeling/immunoprecipitation, we showed that IVA is due to a mutation of IVD. We also demonstrated that there are at least 5 distinct forms of mutant IVD, indicating an extensive molecular heterogeneity. Furthermore, we cloned cDNAs encoding IVD and medium-chain acyl-CoA dehydrogenases. The comparison of their complete primary sequences revealed a high degree of homology, indicating that these enzymes belong to a gene family, the acyl-CoA dehydrogenase family.     

The mitochondrial isovaleryl-coenzyme a dehydrogenase of arabidopsis oxidizes intermediates of leucine and valine catabolism

We recently identified a cDNA encoding a putative isovaleryl-coenzyme A (CoA) dehydrogenase in Arabidopsis (AtIVD). In animals, this homotetrameric enzyme is located in mitochondria and catalyzes the conversion of isovaleryl-CoA to 3-methylcrotonyl-CoA as an intermediate step in the leucine (Leu) catabolic pathway. Expression of AtIVD:smGFP4 fusion proteins in tobacco (Nicotiana tabacum) protoplasts and biochemical studies now demonstrate the in vivo import of the plant isovaleryl-CoA dehydrogenase (IVD) into mitochondria and the enzyme in the matrix of these organelles. Two-dimensional separation of mitochondrial proteins by blue native and SDS-PAGE and size determination of the native and overexpressed proteins suggest homodimers to be the dominant form of the plant IVD. Northern-blot hybridization and studies in transgenic Arabidopsis plants expressing Ativd promoter:gus constructs reveal strong expression of this gene in seedlings and young plants grown in the absence of sucrose, whereas promoter activity in almost all tissues is strongly inhibited by exogeneously added sucrose. Substrate specificity tests with AtIVD expressed in Escherichia coli indicate a strong preference toward isovaleryl-CoA but surprisingly also show considerable activity with isobutyryl-CoA. This strongly indicates a commitment of the enzyme in Leu catabolism, but the activity observed with isobutyryl-CoA also suggests a parallel involvement of the enzyme in the dehydrogenation of intermediates of the valine degradation pathway. Such a dual activity has not been observed with the animal IVD and may suggest a novel connection of the Leu and valine catabolism in plants.     

Characterization of Neurospora crassa Tom40-deficient mutants and effect of specific mutations on Tom40 assembly

The TOM complex (Translocase of the Outer mitochondrial Membrane) is responsible for the recognition of mitochondrial preproteins synthesized in the cytosol and for their translocation across or into the outer mitochondrial membrane. Tom40 is the major component of the TOM complex and forms the translocation pore. We have created a tom40 mutant of Neurospora crassa and have demonstrated that the gene is essential for the viability of the organism. Mitochondria with reduced levels of Tom40 were deficient for import of mitochondrial preproteins and contained reduced levels of the TOM complex components Tom22 and Tom6, suggesting that the import and/or stability of these proteins is dependent on the presence of Tom40. Mutant Tom40 preproteins were analyzed for their ability to be assembled into the TOM complex. In vitro import assays revealed that conserved regions near the N terminus (residues 51-60) and the C terminus (residues 321-323) of the 349-amino acid protein were required for assembly beyond a 250-kDa intermediate form. Mutant strains expressing Tom40 with residues 51-60 deleted were viable but exhibited growth defects. Slow growing mutants expressing Tom40, where residues 321-323 were changed to Ala residues, were isolated but showed TOM complex defects, whereas strains in which residues 321-323 were deleted could not be isolated. Analysis of the assembly of mutant Tom40 precursors in vitro supported a previous model in which Tom40 precursors progress from the 250-kDa intermediate to a 100-kDa form and then assemble into the 400-kDa TOM complex. Surprisingly, when wild type mitochondria containing Tom40 precursors arrested at the 250-kDa intermediate were treated with sodium carbonate, further assembly of intermediates into the TOM complex occurred, suggesting that disruption of protein-protein interactions may facilitate assembly. Import of wild type Tom40 precursor into mitochondria containing a mutant Tom40 lacking residues 40-48 revealed an alternate assembly pathway and demonstrated that the N-terminal region of pre-existing Tom40 molecules in the TOM complex plays a role in the assembly of incoming Tom40 molecules.     

Exon skipping in IVD RNA processing in isovaleric acidemia caused by point mutations in the coding region of the IVD gene

Isovaleric acidemia (IVA) is a recessive disorder caused by a deficiency of isovaleryl-CoA dehydrogenase (IVD). We have reported elsewhere nine point mutations in the IVD gene in fibroblasts of patients with IVA, which lead to abnormalities in IVD protein processing and activity. In this report, we describe eight IVD gene mutations identified in seven IVA patients that result in abnormal splicing of IVD RNA. Four mutations in the coding region lead to aberrantly spliced mRNA species in patient fibroblasts. Three of these are amino acid altering point mutations, whereas one is a single-base insertion that leads to a shift in the reading frame of the mRNA. Two of the coding mutations strengthen pre-existing cryptic splice acceptors adjacent to the natural splice junctions and apparently interfere with exon recognition, resulting in exon skipping. This mechanism for missplicing has not been reported elsewhere. Four other mutations alter either the conserved gt or ag dinucleotide splice sites in the IVD gene. Exon skipping and cryptic splicing were confirmed by transfection of these mutations into a Cos-7 cell line model splicing system. Several of the mutations were predicted by individual information analysis to inactivate or significantly weaken adjacent donor or acceptor sites. The high frequency of splicing mutations identified in these patients is unusual, as is the finding of missplicing associated with missense mutations in exons. These results may lead to a better understanding of the phenotypic complexity of IVA, as well as provide insight into those factors important in defining intron/exon boundaries in vivo.     

Purification, characterization and cloning of isovaleryl-CoA dehydrogenase from higher plant mitochondria.

Between the different types of Acyl-CoA dehydrogenases (ACADs), those specific for branched chain acyl-CoA derivatives are involved in the catabolism of amino acids. In mammals, isovaleryl-CoA dehydrogenase (IVD), an enzyme of the leucine catabolic pathway, is a mitochondrial protein, as other acyl-CoA dehydrogenases involved in fatty acid beta-oxidation. In plants, fatty acid beta-oxidation takes place mainly in peroxisomes, and the cellular location of the enzymes involved in the catabolism of branched-chain amino acids had not been definitely assigned. Here, we describe that highly purified potato mitochondria have important IVD activity. The enzyme was partially purified and cDNAs from two different genes were obtained. The partially purified enzyme has enzymatic constant values with respect to isovaleryl-CoA comparable to those of the mammalian enzyme. It is not active towards straight-chain acyl-CoA substrates tested, but significant activity was also found with isobutyryl-CoA, implying an additional role of the enzyme in the catabolism of valine. The present study confirms recent reports that in plants IVD activity resides in mitochondria and opens the way to a more detailed study of amino-acid catabolism in plant development.     

Genetic heterogeneity in propionic acidemia patients with alpha-subunit defects. Identification of five novel mutations, one of them causing instability of the protein

The inherited metabolic disease propionic acidemia (PA) can result from mutations in either of the genes PCCA or PCCB, which encode the alpha and beta subunits, respectively, of the mitochondrial enzyme propionyl CoA-carboxylase. In this work we have analyzed the molecular basis of PCCA gene defects, studying mRNA levels and identifying putative disease causing mutations. A total of 10 different mutations, none predominant, are present in a sample of 24 mutant alleles studied. Five novel mutations are reported here for the first time. A neutral polymorphism and a variant allele present in the general population were also detected. To examine the effect of a point mutation (M348K) involving a highly conserved residue, we have carried out in vitro expression of normal and mutant PCCA cDNA and analyzed the mitochondrial import and stability of the resulting proteins. Both wild-type and mutant proteins were imported into mitochondria and processed into the mature form with similar efficiency, but the mature mutant M348K protein decayed more rapidly than did the wild-type, indicating a reduced stability, which is probably the disease-causing mechanism.     

Precursor protein is readily degraded in mitochondrial matrix space if the leader is not processed by mitochondrial processing peptidase

It is not known why leader peptides are removed by the mitochondrial processing peptidase after import into the matrix space. The leaders of yeast aldehyde dehydrogenase (pALDH) and malate dehydrogenase were mutated so that they would not be processed after import. The recombinant nonprocessed precursor of yeast pALDH possessed a similar specific activity as the corresponding mature form but was much less stable. The nonprocessed pALDH was transformed into a yeast strain missing ALDHs. The transformed yeast grew slowly on ethanol as the sole carbon source showing that the nonprocessed precursor was functional in vivo. Western blot analysis showed that the amount of precursor was 15-20% of that found in cells transformed with the native enzyme. Pulse-chase experiments revealed that the turnover rate for the nonprocessed precursor was greater than that of the mature protein indicating that the nonprocessed precursor could have been degraded. By using carbonyl cyanide m-chlorophenylhydrazone, we showed that the nonprocessed precursor was degraded in the matrix space. The nonprocessed precursor forms of precursor yeast malate dehydrogenase and rat liver pALDH also were degraded in the matrix space of HeLa cell mitochondria faster than their corresponding mature forms. In the presence of o-phenanthroline, an inhibitor of mitochondrial processing peptidase, the wild type precursor was readily degraded in the matrix space. Collectively, this study showed that the precursor form is less stable in the matrix space than is the mature form and provides an explanation for why the leader peptide is removed from the precursors.     

Structural and functional effects of mutations altering the subunit interface of mitochondrial malate dehydrogenase

Among highly conserved residues in eucaryotic mitochondrial malate dehydrogenases are those with roles in maintaining the interactions between identical monomeric subunits that form the dimeric enzymes. The contributions of two of these residues, Asp-43 and His-46, to structural stability and catalytic function were investigated by construction of mutant enzymes containing Asn-43 and Leu-46 substitutions using in vitro mutagenesis of the Saccharomyces cerevisiae gene (MDH1) encoding mitochondrial malate dehydrogenase. The mutant enzymes were expressed in and purified from a yeast strain containing a disruption of the chromosomal MDH1 locus. The enzyme containing the H46L substitution, as compared to the wild type enzyme, exhibits a dramatic shift in the pH profile for catalysis toward an optimum at low pH values. This shift corresponds with an increased stability of the dimeric form of the mutant enzyme, suggesting that His-46 may be the residue responsible for the previously described pH-dependent dissociation of mitochondrial malate dehydrogenase. The D43N substitution results in a mutant enzyme that is essentially inactive in in vitro assays and that tends to aggregate at pH 7.5, the optimal pH for catalysis for the dimeric wild type enzyme.     

Isolation of cDNA clones coding for rat isovaleryl-CoA dehydrogenase and assignment of the gene to human chromosome 15

Rat liver mRNA encoding the cytoplasmic precursor of mitochondrial isovaleryl-CoA dehydrogenase was highly enriched by polysome immunopurification using a polyclonal monospecific antibody. The purified mRNA was used to prepare a plasmid cDNA library which was screened with two oligonucleotide mixtures encoding two peptides in the amino-terminal portion of mature rat isovaleryl-CoA dehydrogenase. Thirty-one overlapping cDNA clones, spanning a region of 2.1 kbp, were isolated and characterized. The cDNA sequence of a 5'-end clone, rIVD-13 (155 bp), predicts a mitochondrial leader peptide of 30 amino acid residues and the first 18 amino acids of the mature protein. These consecutive 18 residues completely matched the amino-terminal peptide determined by automated Edman degradation of the rat enzyme. The leader peptide contains six arginines, has no acidic residues, and is particularly rich in leucine, alanine, and proline residues. Southern blot analysis of DNAs from human-rodent somatic cell hybrids with an isolated rat cDNA (2 kbp) assigned the isovaleryl-CoA dehydrogenase gene to the long arm of chromosome 15, region q14----qter. The chromosomal assignment was confirmed and further refined to bands q14----q15 by in situ hybridization of the probe to human metaphase cells. This location differs from that of the gene for medium-chain acyl-CoA dehydrogenase, a closely related enzyme, which has been previously assigned to chromosome 1.     

High-level expression of an altered cDNA encoding human isovaleryl-CoA dehydrogenase in Escherichia coli

Isovaleryl-CoA dehydrogenase (IVD) catalyzes the conversion of isovaleryl-CoA to 3-methylcrotonyl-CoA in the leucine catabolism pathway. The cDNA encoding the mature human IVD polypeptide was cloned in a prokaryotic expression vector, but the level of expression in Escherichia coli was extremely low and attempts to purify the enzyme to homogeneity were unsuccessful. To enhance expression, the nucleotide sequence of 22 codons within the 111-bp region at the 5′-end of the cDNA was altered to accommodate E. coli codon usage without altering the amino-acid coding sequence. The altered IVD cDNA was synthesized by PCR, using a primer containing the desired modifications. Following overnight induction of the E. coli transformed with this cDNA, the enzyme was purified to homogeneity using diethylaminoethyl agarose and high-pressure ceramic hydroxyapatite resins. IVD activity was increased 165-fold in the crude extract of cells containing the modified cDNA, as compared to that containing the wild-type cDNA.     

Biosynthesis and processing of renal mitochondrial glutaminase in cultured proximal tubular epithelial cells and in isolated mitochondria

Primary cultures of rat renal proximal tubular epithelial cells were used to characterize the biosynthesis and processing of the mitochondrial glutaminase. When the cells were labeled with [35S]methionine in the presence of 20 microM carbonylcyanide m-chlorophenylhydrazone, only a 72-kDa peptide, which co-migrates with the primary translation product of the glutaminase mRNA, was immunoprecipitated. At lower concentrations of carbonylcyanide m-chlorophenylhydrazone, the 68- and 65-kDa peptides that are characteristic of the mature glutaminase and a 71-kDa peptide were synthesized. Pulse-chase experiments suggest that the 72-kDa cytosolic precursor could be quantitatively chased to generate the mature mitochondrial species. The observed kinetics indicate that the 71-kDa species is an intermediate in the import pathway. In addition, the 65-kDa glutaminase peptide was synthesized more rapidly than the 68-kDa peptide, and the two peptides were produced in a final ratio of 3:1, respectively. These results suggest that one subunit of the tetrameric glutaminase may be subject to covalent modification. In vitro processing was also characterized by incubating isolated rat liver mitochondria with the glutaminase precursor that was produced by in vitro translation of acidotic rat renal poly(A+) RNA. This system produced an identical sequence of processing reactions. The in vitro formation of the 71-kDa intermediate required a transmembrane potential. Both the intermediate and the mature forms of the glutaminase were recovered in the mitochondria and were resistant to trypsin digestion. Thus, the glutaminase precursor is rapidly translocated across the inner mitochondrial membrane and initially processed to yield an intermediate. The intermediate is subsequently processed to yield the two peptides that constitute the mature enzyme.     

Convergent evolution of a 2-methylbutyryl-CoA dehydrogenase from isovaleryl-CoA dehydrogenase in Solanum tuberosum

The potato cDNAs Solanum tuberosum isovaleryl-CoA dehydrogenases 1 and 2 (St-IVD1 and St-IVD2) encode proteins that are 84% identical to each other and 65 and 64% identical to human IVD, respectively. St-IVD2 protein was previously partially purified from potato tubers and confirmed to be an IVD. The function of St-IVD1 is unknown. In these experiments, both proteins were expressed in Escherichia coli and purified as intact homotetramers. The substrate preference profile of the St-IVD2 protein was similar to that of human IVD. However, recombinant St-IVD1 had maximal activity with 2-methylbutyryl-CoA, which in humans is dehydrogenated by short/branched-chain acyl-CoA dehydrogenase (SBCAD). Whereas molecular modeling predicts that the 2-methylbutyryl-CoA dehydrogenase (2MBCD) and IVD substrate binding pockets are nearly identical, 2MBCD has amino acid substitutions at five residues that are invariant among all of the known and putative IVDs. Site-directed mutagenesis was used to match the human IVD active site with that of potato 2MBCD. The resulting mutant IVD had detectable activity with 2-methylbutyryl-CoA and no activity with isovaleryl-CoA. The 2MBCD active site was compared with that of human SBCAD using molecular modeling. Residues Met-361 and Ala-365 of 2MBCD appear to partially substitute for the function of Tyr-380 in human SBCAD, binding the methyl branch linked to C2 of 2-methylbutyryl-CoA, whereas residues Val-88, Val-92, and Val-96 appear to bind the distal C4 methyl group. The presence of a 2MBCD in potato that is highly homologous to IVD is an example of convergent evolution within the acyl-CoA dehydrogenase family, leading to the independent occurrence of two enzymes (SBCAD and 2MBCD) specific for 2-methylbutyryl-CoA.     

In vivo mitochondrial import. A comparison of leader sequence charge and structural relationships with the in vitro model resulting in evidence for co-translational import

The positive charges and structural properties of the mitochondrial leader sequence of aldehyde dehydrogenase have been extensively studied in vitro. The results of these studies showed that increasing the helicity of this leader would compensate for reduced import from positive charge substitutions of arginine with glutamine or the insertion of negative charged residues made in the native leader. In this in vivo study, utilizing the green fluorescent protein (GFP) as a passenger protein, import results showed the opposite effect with respect to helicity, but the results from mutations made within the native leader sequence were consistent between the in vitro and in vivo experiments. Leader mutations that reduced the efficiency of import resulted in a cytosolic accumulation of a truncated GFP chimera that was fluorescent but devoid of a mitochondrial leader. The native leader efficiently imported before GFP could achieve a stable, import-incompetent structure, suggesting that import was coupled with translation. As a test for a co-translational mechanism, a chimera of GFP that contained the native leader of aldehyde dehydrogenase attached at the N terminus and a C-terminal endoplasmic reticulum targeting signal attached to the C terminus of GFP was constructed. This chimera was localized exclusively to mitochondria. The import result with the dual signal chimera provides support for a co-translational mitochondrial import pathway.     

Molecular defect of isovaleryl-CoA dehydrogenase in the skunk mutant of silkworm, Bombyx mori

The isovaleric acid-emanating silkworm mutant skunk (sku) was first studied over 30?years ago because of its unusual odour and prepupal lethality. Here, we report the identification and characterization of the gene responsible for the sku mutant. Because of its specific features and symptoms similar to human isovaleryl-CoA dehydrogenase (IVD) deficiency, also known as isovaleric acidaemia, IVD dysfunction in silkworms was predicted to be responsible for the phenotype of the sku mutant. Linkage analysis revealed that the silkworm IVD gene (BmIVD) was closely linked to the odorous phenotype as expected, and a single amino acid substitution (G376V) was found in BmIVD of the sku mutant. To investigate the effect of the G376V substitution on BmIVD function, wild-type and sku-type recombinants were constructed with a baculovirus expression system and the subsequent enzyme activity of sku-type BmIVD was shown to be significantly reduced compared with that of wild-type BmIVD. Molecular modelling suggested that this reduction in the enzyme activity may be due to negative effects of G376V mutation on FAD-binding or on monomer-monomer interactions. These observations strongly suggest that BmIVD is responsible for the sku locus and that the molecular defect in BmIVD causes the characteristic smell and prepupal lethality of the sku mutant. To our knowledge, this is, aside from humans, the first characterization of IVD deficiency in metazoa. Considering that IVD acts in the third step of leucine degradation and the sku mutant accumulates branched-chain amino acids in haemolymph, this mutant may be useful in the investigation of unique branched-chain amino acid catabolism in insects.     

Comparative studies of Acyl-CoA dehydrogenases for monomethyl branched chain substrates in amino acid metabolism

Short/branched chain acyl-CoA dehydrogenase (SBCAD), isovaleryl-CoA dehydrogenase (IVD), and isobutyryl-CoA dehydrogenase (IBD) are involved in metabolism of isoleucine, leucine, and valine, respectively. These three enzymes all belong to acyl-CoA dehydrogenase (ACD) family, and catalyze the dehydrogenation of monomethyl branched-chain fatty acid (mmBCFA) thioester derivatives. In the present work, the catalytic properties of rat SBCAD, IVD, and IBD, including their substrate specificity, isomerase activity, and enzyme inhibition, were comparatively studied. Our results indicated that SBCAD has its catalytic properties relatively similar to those of straight-chain acyl-CoA dehydrogenases in terms of their isomerase activity and enzyme inhibition, while IVD and IBD are different. IVD has relatively broader substrate specificity than those of the other two enzymes in accommodating various substrate analogs. The present study increased our understanding for the metabolism of monomethyl branched-chain fatty acids (mmBCFAs) and branched-chain amino acids (BCAAs), which should also be useful for selective control of a particular reaction through the design of specific inhibitors.