Biochemical Studies on Recombinant Human Isobutyryl-CoA Dehydrogenase (original) (raw)
Related papers
Biochemistry, 1997
Isovaleryl-CoA dehydrogenase (IVD) belongs to an important flavoprotein family of acyl-CoA dehydrogenases that catalyze the R,-dehydrogenation of their various thioester substrates. Although enzymes from this family share similar sequences, catalytic mechanisms, and structural properties, the position of the catalytic base in the primary sequence is not conserved. E376 has been confirmed to be the catalytic base in medium-chain (MCAD) and short-chain acyl-CoA dehydrogenases and is conserved in all members of the acyl-CoA dehydrogenase family except for IVD and long-chain acyl-CoA dehydrogenase. To understand this dichotomy and to gain a better understanding of the factors important in determining substrate specificity in this enzyme family, the three-dimensional structure of human IVD has been determined. Human IVD expressed in Escherichia coli crystallizes in the orthorhombic space group P2 1 2 1 2 1 with unit cell parameters a) 94.0 Å, b) 97.7 Å, and c) 181.7 Å. The structure of IVD was solved at 2.6 Å resolution by the molecular replacement method and was refined to an R-factor of 20.7% with an R free of 28.8%. The overall polypeptide fold of IVD is similar to that of other members of this family for which structural data are available. The tightly bound ligand found in the active site of the structure of IVD is consistent with that of CoA persulfide. The identity of the catalytic base was confirmed to be E254, in agreement with previous molecular modeling and mutagenesis studies. The location of the catalytic residue together with a glycine at position 374, which is a tyrosine in all other members of the acyl-CoA dehydrogenase family, is important for conferring branched-chain substrate specificity to IVD.
Identification of the Active Site Catalytic Residue in Human Isovaleryl-CoA Dehydrogenase
Biochemistry, 1995
Isovaleryl-CoA dehydrogenase (IVD) is a homotetrameric flavoenzyme which catalyzes the conversion of isovaleryl-CoA to 3-methylcrotonyl-CoA. E376 of pig medium chain acyl-CoA dehydrogenase (MCAD), a homologous enzyme, has been identified as the active site catalytic residue. Amino acid sequence alignment shows that A375 is the corresponding residue in human IVD. Using the atomic coordinates determined for MCAD, molecular modeling suggests that E254 is the substituting catalytic residue in IVD. To substantiate the importance of this residue for enzyme function, cDNAs for the wildtype human IVD and E254G, E254D, E254Q, and E254G/A375E mutant IVDs were constructed and cloned into a prokaryotic expression vector. The proteins were synthesized in Escherichia coli and purified, and their properties were examined. The catalytic activity of the recombinant wild-type IVD was the highest in the presence of isovaleryl-CoA, and its UV/visible light spectrum in the presence of isovaleryl-CoA showed quenching of its characteristic absorption in the 445-nm region and appearance of absorption at 600 nm. 'The E254G and E254Q mutant IVDs had no detectable enzymatic activity, and isovaleryl-CoA did not induce quenching of the absorption in the 445-nm region or the appearance of absorption at 600 nm. The E254D mutant IVD had residual activity for isovaleryl-CoA, and its spectrum was altered compared to that of the wild type. The E254G/A375E mutant IVD exhibited catalytic activity toward isovaleryl-CoA, and its spectrum in the absence or presence of the substrate was similar to that of the wild-type IVD. These results suggest that E254 of IVD is in close proximity to the bound FAD and support the hypothesis that E254 is the active site catalytic residue. Isovaleryl-CoA dehydrogenase (IVD;' EC 1.3.99.10) catalyzes the conversion of isovaleryl-CoA to 3-methylcrotonyl-CoA in the leucine catabolism pathway. Deficiency of the enzyme in humans is responsible for isovaleric acidemia, a serious metabolic disorder (
Identification of isobutyryl-CoA dehydrogenase and its deficiency in humans
Molecular Genetics and Metabolism, 2002
The acyl-CoA dehydrogenases (ACDs) are a family of related enzymes that catalyze the a,b-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 (lM À1 s À1 ) 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. Ó
Kinetic and spectral properties of isovaleryl-CoA dehydrogenase and interaction with ligands
Biochimie, 2015
Isovaleryl-CoA dehydrogenase (IVD) catalyzes the conversion of isovaleryl-CoA to 3methylcrotonyl-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 K m and catalytic efficiency (k cat /K m) for isovaleryl-CoA were 1.0 µM and 4.3 × 10 6 •M −1 •sec −1 per monomer, respectively, and its K m 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 (K D 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 K i of 1.5 mM. Keywords acyl-CoA dehydrogenase; electron-transferring flavoprotein Isovaleryl-CoA dehydrogenase (IVD; 1 EC 1.3.99.10) is an intramitochondrial homotetrameric flavoenzyme in the leucine catabolism pathway that catalyzes the α,β
Biochemistry, 1995
We offer a large scale purification procedure for the recombinant human liver medium-chain acyl-CoA dehydrogenase (HMCAD). This procedure routinely yields 100-150 mg of homogeneous preparation of the enzyme from 80 L of the Escherichia coli host cells. A comparative investigation of kinetic properties of the human liver and pig kidney enzymes revealed that, except for a few minor differences, both of these enzymes are nearly identical. We undertook detailed kinetic and thermodynamic investigations for the interaction of HMCAD-FAD with three Cg-CoA molecules (viz., octanoyl-CoA, 2-octenoyl-CoA, and 2-octynoyl-CoA), which differ with respect to the extent of unsaturation at the a-P carbon centers; octanoyl-CoA and 2-octenoyl-CoA serve as the substrate and product of the enzyme, respectively, whereas 2-octynoyl-CoA is known to inactivate the enzyme. Our experimental results demonstrate that all three Cg-CoA molecules first interact with HMCAD-FAD to form corresponding Michaelis complexes, followed by two subsequent isomerization reactions. The latter accompany either subtle changes in the electronic structures of the individual components (in case of 2-octenoyl-CoA and 2-octynoyl-CoA ligands), or a near-complete reduction of the enzyme-bound flavin (in case of octanoyl-CoA). The rate and equilibrium constants intrinsic to the above microscopic steps exhibit marked similarity with different Cg-CoA molecules. However, the electronic structural changes accompanying the 2-octynoyl-CoA-dependent inactivation of the enzyme is 3-4 orders of magnitude slower than the above isomerization reactions. Hence, the octanoyl-CoA-dependent reductive half-reaction and the 2-octynoyl-CoA-dependent covalent modification of the enzyme occur during entirely different microscopic steps. Arguments are presented that the origin of the above difference lies in the protein conformation-dependent orientation of Glu-376 in the vicinity of the Cg-CoA binding site. In a series of publications over the past four years, we have elaborated on several mechanistic aspects of the medium-chain acyl-CoA dehydrogenase (MCAD)-cataly zed' "dehydrogenase" and "oxidase" reactions (
The purification and properties of ox liver short-chain acyl-CoA dehydrogenase
The Biochemical journal, 1984
The FAD-containing short-chain acyl-CoA dehydrogenase was purified from ox liver mitochondria by using (NH4)2SO4 fractionation, DEAE-Sephadex A-50 and chromatofocusing on PBE 94 resin. The enzyme is a tetramer, with a native Mr of approx. 162 000 and a subunit Mr of 41 000. Short-chain acyl-CoA dehydrogenases are usually isolated in a green form. The chromatofocusing step in the purification presented here partially resolved the enzyme into a green form and a yellow form. In the dye-mediated assay system, the enzyme exhibited optimal activity towards 50 microM-butyryl-CoA at pH 7.1. Kinetic parameters were also determined for a number of other straight-chain acyl-CoA substrates. The u.v.- and visible-absorption characteristics of the native forms of the enzyme are described, together with complexes formed by addition of butyryl-CoA, acetoacetyl-CoA and CoA persulphide.
Arginine 387 of Human Isovaleryl-CoA Dehydrogenase Plays a Crucial Role in Substrate/Product Binding
Molecular Genetics and Metabolism, 2001
Isovaleryl-CoA dehydrogenase (IVD) is a homotetrameric flavoenzyme, which catalyzes the conversion of isovaleryl-CoA to 3-methylcrotonyl-CoA and transfers electrons to the electron-transferring flavoprotein, and is a member of the acyl-CoA dehydrogenase (ACD) enzyme family. Human IVD crystal structure with a bound substrate analogue shows the guanidino group of Arg387, a conserved residue among other members of the ACD enzyme family, juxtaposed to a phosphate oxygen of the 4-phosphopantothiene moiety of the substrate analogue. Site-directed mutagenesis was used to investigate the role of Arg387 in substrate binding and enzyme function. Replacing this residue with Lys, Ala, Gln, or Glu resulted in stable proteins. Spectrophotometric substrate binding assays indicated that the Arg387Lys mutant was able to form the charge-transfer complex intermediate with similar efficiency to wild type, while the rest of the mutants were significantly less able to properly form this intermediate. However, the K m of the isovaleryl-CoA for the Arg387Lys mutant was 20.3 compared to 1.5 M for the wild type. The K m for the rest of the mutants were 75.6, 195, and 550 M, respectively. The catalytic efficiency per mole of FAD was 20.3, 3.3, 2.0, and 0.34 for the mutants, respectively, compared to 260 M ؊1 min ؊1 for the wild type. These results substantiate the important role of Arg387 in anchoring the substrate, and are consistent with the hypothesis that residues distant from the active site are important for stabilizing the en-zyme:substrate/product complex, and could play an important role in the mechanism of the enzymecatalyzed reaction.
Biochemistry, 1988
Thia-and oxaoctanoyl-CoA derivatives (substituted at the C-3 and C-4 positions) have been synthesized to probe the reductive half-reaction in the medium-chain acyl-CoA dehydrogenase from pig kidney. 3-Thiaoctanoyl-CoA binds to this flavoenzyme, forming an intense, stable, long-wavelength band (at 804 nm; extinction coefficient = 8.7 mM-' cm-' at p H 7.6). The intensity of this band increases about 20% from p H 6.0 to p H 8.8. This long-wavelength species probably represents a charge-transfer complex between bound acyl enolate as the donor and oxidized flavin adenine dinucleotide as the acceptor. Thus, the enzyme catalyzes a-proton exchange, and no long-wavelength bands are seen with 3-thiaoctyl-CoA (where the carbonyl moiety is replaced by a methylene group). 3-Oxaoctanoyl-CoA binds comparatively weakly to the dehydrogenase, with a long-wavelength band at 780 nm which is both less intense and less stable than the corresponding thia analogue. These data suggest that the enzyme can accomplish a-proton abstraction from certain weakly acidic acyl-CoA derivatives, without concerted transfer of a hydride equivalent to the flavin. 4-Thiaoctanoyl-CoA is dehydrogenated in the standard assay 1.5-fold faster than octanoyl-CoA. Titrations of the medium-chain dehydrogenase with the 4-thia derivative resemble those obtained with octanoyl-CoA, except for the contribution of the strongly absorbing 4-thia-trans-2-octenoyl-CoA product.
Biochemistry, 1998
Isovaleryl-CoA dehydrogenase (IVD) is a homotetrameric mitochondrial flavoenzyme which catalyzes the conversion of isovaleryl-CoA to 3-methylcrotonyl-CoA. PCR of IVD genomic and complementary DNA was used to identify mutations occurring in patients with deficiencies in IVD activity. Western blotting, in vitro mitochondrial import, prokaryotic expression, and kinetic studies of IVD mutants were conducted to characterize the molecular defects caused by the amino acid replacements. Mutations leading to Arg21Pro, Asp40Asn, Ala282Val, Cys328Arg, Val342Ala, Arg363Cys, and Arg382Leu replacements were identified. Western blotting of fibroblast extracts and/or in vitro mitochondrial import experiments indicate that the seven precursor IVD mutant peptides, and a previously identified IVD Leu13Pro mutant, are synthesized and imported into mitochondria. While the IVD Leu13Pro, Arg21Pro, and Cys328Arg mutant peptides are rapidly degraded following mitochondrial import, the other mutant peptides exhibit greater mitochondrial stability, though less than the wild-type enzyme. Active IVD Ala282Val, Val342Ala, Arg363Cys, and Arg382Leu mutants were less stable than wild type when produced in Escherichia coli. The K m values of purified IVD Ala282Val, Val342Ala, and Arg382Leu mutants are 27.0, 2.8, and 6.9 µM isovaleryl-CoA, respectively, compared to 3.1 µM for the wild type, using the electron-transfer flavoprotein (ETF) fluorescence quenching assay. The catalytic efficiency per mole of FAD content of these three mutants is 4.8, 17.0, and 17.0 µM-1 •min-1 , respectively, compared to 170 µM-1 •min-1 for wild type.
Archives of Biochemistry and Biophysics, 1996
The acyl-CoA dehydrogenases (ACDs) 3 are a family The acyl-CoA dehydrogenases are a family of re-of related enzymes which catalyze the a,b-dehydrogelated enzymes which catalyze the a,b-dehydrogena-nation of acyl-CoA esters, transferring electrons to election of acyl-CoA esters, transferring electrons to tron-transferring flavoprotein [ETF, (1-6)]. Deficiencelectron-transferring flavoprotein. A cDNA for huies of these enzymes are important causes of human man short/branched chain acyl-CoA dehydrogenase disease (7, 8). Biochemical and immunological studies has recently been cloned, and it has been suggested have identified at least six distinct members of this that this enzyme represents the human homolog for enzyme family, each with a narrow substrate specificity the previously reported 2-methyl branched chain (2-5, 9, 10). Very long, long, medium, and short chain acyl-CoA dehydrogenase purified from rat liver. We acyl-CoA dehydrogenases (VLCAD, LCAD, MCAD, and now report the cloning and expression of rat short/ SCAD) catalyze the first step in the b-oxidation of branched chain acyl-CoA dehydrogenase and charstraight chain fatty acids with substrate optima of acterization of its substrate specificity. The rat en-16, 16, 8, and 4 carbons, respectively (2, 5, 9, 10). zyme is more active toward longer carbon side Isovaleryl-CoA dehydrogenase (IVD) and a 2-methyl chains than its human counterpart, while the human branched chain acyl-CoA dehydrogenase (2-meBCAD) enzyme can utilize substrates with longer primary catalyze the third step in leucine and isoleucine/valine carbon chains. In addition, short/branched chain metabolism, respectively (2-4). A purified rat 2-meBacyl-CoA dehydrogenase can utilize valproyl-CoA as CAD has been shown to have 8.4% activity with vala substrate. Northern blotting of mRNA shows ubiqproyl-CoA compared with its optimum substrate (S)-2uitous tissue expression of both the rat and human methylbutyryl-CoA (11). We have recently cloned and enzyme. Further study of these enzymes will be helpcharacterized a cDNA for human short/branched chain ful in understanding structure/function relationacyl-CoA dehydrogenase (SBCAD) and have suggested ships in this gene family.