Physiological Perspectives on the Use of Triheptanoin as Anaplerotic Therapy for Long Chain Fatty Acid Oxidation Disorders.

Inborn errors of mitochondrial fatty acid oxidation (FAO) comprise the most common group of disorders identified through expanded newborn screening mandated in all 50 states in the United States, affecting 1:10,000 newborns. While some of the morbidity in FAO disorders (FAODs) can be reduced if identified through screening, a significant gap remains between the ability to diagnose these disorders and the ability to treat them. At least 25 enzymes and specific transport proteins are responsible for carrying out the steps of mitochondrial fatty acid metabolism, with at least 22 associated genetic disorders. Common symptoms in long chain FAODs (LC-FAODs) in the first week of life include cardiac arrhythmias, hypoglycemia, and sudden death. Symptoms later in infancy and early childhood may relate to the liver or cardiac or skeletal muscle dysfunction, and include fasting or stress-related hypoketotic hypoglycemia or Reye-like syndrome, conduction abnormalities, arrhythmias, dilated or hypertrophic cardiomyopathy, and muscle weakness or fasting- and exercise-induced rhabdomyolysis. In adolescent or adult-onset disease, muscular symptoms, including rhabdomyolysis, and cardiomyopathy predominate. Unfortunately, progress in developing better therapeutic strategies has been slow and incremental. Supplementation with medium chain triglyceride (MCT; most often a mixture of C8-12 fatty acids containing triglycerides) oil provides a fat source that can be utilized by patients with long chain defects, but does not eliminate symptoms. Three mitochondrial metabolic pathways are required for efficient energy production in eukaryotic cells: oxidative phosphorylation (OXPHOS), FAO, and the tricarboxylic (TCA) cycle, also called the Krebs cycle. Cell and mouse studies have identified a deficiency in TCA cycle intermediates in LC-FAODs, thought to be due to a depletion of odd chain carbon compounds in patients treated with a predominantly MCT fat source. Triheptanoin (triheptanoyl glycerol; UX007, Ultragenyx Pharmaceuticals) is chemically composed of three heptanoate (seven carbon fatty acid) molecules linked to glycerol through ester bonds that has the potential to replete TCA cycle intermediates through production of both acetyl-CoA and propionyl-CoA through medium chain FAO. Compassionate use, retrospective, and recently completed prospective studies demonstrate significant reduction of hypoglycemic events and improved cardiac function in LC-FAOD patients, but a less dramatic effect on muscle symptoms.

Kinetic and spectral properties of isovaleryl-CoA dehydrogenase and interaction with ligands.

Isovaleryl-CoA dehydrogenase (IVD) catalyzes the conversion of isovaleryl-CoA to 3-methylcrotonyl-CoA and the transfer of electrons to the electron transfer flavoprotein (ETF). Recombinant human IVD purifies with bound CoA-persulfide. A modified purification protocol was developed to isolate IVD without bound CoA-persulfide and to protect the protein thiols from oxidation. The CoA-persulfide-free IVD specific activity was 112.5 µmol porcine ETF min(-)(1) mg(-)(1), which was ∼20-fold higher than that of its CoA-persulfide bound form. The Km and catalytic efficiency (kcat/Km) for isovaleryl-CoA were 1.0 µM and 4.3 × 10(6) M(-1) s(-1) per monomer, respectively, and its Km for ETF was 2.0 µM. Anaerobic titration of isovaleryl-CoA into an IVD solution resulted in a stable blue complex with increased absorbance at 310 nm, decreased absorbance at 373 and 447 nm, and the appearance of the charge transfer complex band at 584 nm. The apparent dissociation constant (KDapp) determined spectrally for isovaleryl-CoA was 0.54 µM. Isovaleryl-CoA, acetoacetyl-CoA, methylenecyclopropyl-acetyl-CoA, and ETF induced CD spectral changes at the 250-500 nm region while isobutyryl-CoA did not, suggesting conformational changes occur at the flavin ring that are ligand specific. Replacement of the IVD Trp166 with a Phe did not block IVD interaction with ETF, indicating that its indole ring is not essential for electron transfer to ETF. A twelve amino acid synthetic peptide that matches the sequence of the ETF docking peptide competitively inhibited the enzyme reaction when ETF was used as the electron acceptor with a Ki of 1.5 mM.

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.

Thermodynamic basis of electron transfer in dihydroorotate dehydrogenase B from Lactococcus lactis: analysis by potentiometry, EPR spectroscopy, and ENDOR spectroscopy.

Dihydroorotate dehydrogenase B (DHODB) is a complex iron-sulfur flavoprotein that catalyzes the conversion of dihydroorotate to orotate and the reduction of NAD(+). The enzyme is a dimer of heterodimers containing an FMN, an FAD, and a 2Fe-2S center. UV-visible, EPR, and ENDOR spectroscopies have been used to determine the reduction potentials of the flavins and the 2Fe-2S center and to characterize radicals and their interactions. Reductive titration using dithionite indicates a five-electron capacity for DHODB. The midpoint reduction potential of the 2Fe-2S center (-212 +/- 3 mV) was determined from analysis of absorption data at 540 nm, where absorption contributions from the two flavins are small. The midpoint reduction potentials of the oxidized/semiquinone (E(1)) and semiquinone/hydroquinone (E(2)) couples for the FMN (E(1) = -301 +/- 6 mV; E(2) = -252 +/- 8 mV) and FAD (E(1) = -312 +/- 6 mV; E(2) = -297 +/- 5 mV) were determined from analysis of spectral changes at 630 nm. Corresponding values for the midpoint reduction potentials for FMN (E(1) = -298 +/- 4 mV; E(2) = -259 +/- 5 mV) in the isolated catalytic subunit (subunit D, which lacks the 2Fe-2S center and FAD) are consistent with the values determined for the FMN couples in DHODB. During reductive titration of DHODB, small amounts of the neutral blue semiquinone are observed at approximately 630 nm, consistent with the measured midpoint reduction potentials of the flavins. An ENDOR spectrum of substrate-reduced DHODB identifies hyperfine couplings to proton nuclei similar to those recorded for the blue semiquinone of free flavins in aqueous solution, thus confirming the presence of this species in DHODB. Spectral features observed during EPR spectroscopy of dithionite-reduced DHODB are consistent with the midpoint reduction potentials determined using UV-visible spectroscopy and further identify an unusual EPR signal with very small rhombic anisotropy and g values of 2.02, 1.99, and 1.96. This unusual signal is assigned to the formation of a spin interacting state between the FMN semiquinone species and the reduced 2Fe-2S center. Reduction of DHODB using an excess of NADH or dihydroorotate produces EPR spectra that are distinct from those produced by dithionite. From potentiometric studies, the reduction of the 2Fe-2S center and the reduction of the FMN occur concomitantly. The study provides a detailed thermodynamic framework for electron transfer in this complex iron-sulfur flavoprotein.

Identification of isobutyryl-CoA dehydrogenase and its deficiency in humans.

The acyl-CoA dehydrogenases (ACDs) are a family of related enzymes that catalyze the alpha,beta-dehydrogenation of acyl-CoA esters. Two homologues active in branched chain amino acid metabolism have previously been identified. We have used expression in Escherichia coli to produce a previously uncharacterized ACD-like sequence (ACAD8) and define its substrate specificity. Purified recombinant enzyme had a k(cat)/K(m) of 0.8, 0.23, and 0.04 (microM(-1)s(-1)) with isobutyryl-CoA, (S) 2-methylbutyryl-CoA, and n-propionyl-CoA, respectively, as substrates. Thus, this enzyme is an isobutyryl-CoA dehydrogenase. A single patient has previously been described whose fibroblasts exhibit a specific deficit in the oxidation of valine. Amplified ACAD8 cDNA made from patient fibroblast mRNA was homozygous for a single nucleotide change (905G>A) in the ACAD8 coding region compared to the sequence from control cells. This encodes an Arg302Gln substitution in the full-length protein (position 280 in the mature protein), a position predicted by molecular modeling to be important in subunit interactions. The mutant enzyme was stable but inactive when expressed in E. coli. It was also stable and appropriately targeted to mitochondria, but inactive when expressed in mammalian cells. These data confirm further the presence of a separated ACD in humans specific to valine catabolism (isobutyryl-CoA dehydrogenase, IBDH), along with the first enzymatic and molecular confirmation of a deficiency of this enzyme in a patient.