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

Abstract

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 × 106•M−1•sec−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 (KD app) determined spectrally for isovaleryl-CoA was 0.54 µM. Isovaleryl-CoA, acetoacetyl-CoA, methylenecyclopropylacetyl-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.

MoIVD-Mediated Leucine Catabolism Is Required for Vegetative Growth, Conidiation and Full Virulence of the Rice Blast Fungus Magnaporthe oryzae

“…The IVD mutation destroys leucine catabolism at the third step, involving the conversion of isovaleryl-CoA to β-methylcrotonyl-CoA. An accumulation of leucine has not been observed in IVD mutants ( Mohsen et al, 1998 ; Mohsen and Vockley, 2015 ) and we also did not find an obvious high level of leucine in Δ moivd-3 ( Supplementary Figure S9 ), but rather a high level of isovaleric acid was present ( Figure 6A and Supplementary Figure S4 ). These findings suggest that the IVD mutation led to a block of leucine catabolism at the third step, and that accumulated isovaleryl-CoA is converted into isovaleric acid that is not further degraded.…”

“…Leucine is one of the three branched amino acids, and its catabolism provides key intermediate metabolites—acetyl-CoA and ATP—for other metabolic processes ( Lei et al, 2012 ; Arany and Neinast, 2018 ). Isovaleryl-CoA dehydrogenase (IVD) is an enzyme catalyzing the third reaction of the leucine catabolism pathway by conversion of isovaleryl-CoA to β-methylcrotonyl-CoA, and simultaneously electrons transfer to the respiratory chain for ATP production via electron-transferring flavoprotein (ETF) ( Mohsen et al, 1998 ; Zhang et al, 2006 ; Mohsen and Vockley, 2015 ). As a flavoprotein with flavin adenine dinucleotide (FAD) as the cofactor, IVD catalyzes α,β-dehydrogenation and removal of one hydrogen as a proton from the branched-chain substrate, isovaleryl-CoA ( Tiffany et al, 1997 ).…”

Differential impact of amino acids on OXPHOS system activity following carbohydrate starvation in Arabidopsis cell suspensions

“…It is reasonable to assume that this result is most likely related to protein degradation and recycling of amino acids (Araújo et al , Hildebrandt et al ) and thus free BCAA released from protein degradation is used for alternative respiration. Although protein degradation might release only a relatively small amount of free amino acids it is possible that this could be enough to induce IVDH activity given that it has a low K m value of about 1.0 μ M (Weigelt et al , Mohsen and Vockley ). IVDH activity was not altered in presence of lysine even though it has been described as an electron donor to the ETF/ETFQO system (Araújo et al ).…”

Inclusão Da Midia Digital Como Tecnologia Empreendedora No Cuidado Materno Infantil

“…A acidemia isovalérica é uma doença genética de herança autossômica recessiva na qual distúrbios relacionados a um único gene, o isovaleryl-CoA dehydrogenase (IVD), ocasionam a perda da função de uma única proteína, a enzima isovaleril CoA desidrogenase (uma flavoenzima homotetramérica mitocondrial), resultando em concentrações anormais de metabólitos vinculados na sequência de reação (MOHSEN;VOCKLEY, 2015).…”

“…Esse desequilíbrio das substâncias é detectável na urina, plasma e sangue, especialmente em programas de triagem neonatal (MOHSEN;VOCKLEY, 2015).…”