Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:1.3.99.3 (acyl-CoA dehydrogenase)
 1,425 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Flavin adenine dinucleotide (FAD) is a common cofactor in redox proteins, and its reduction potentials are controlled by the protein environment. This regulation is mainly responsible for the versatile catalytic functions of flavoenzymes. In this article, we report computations of the reduction potentials of FAD in medium-chain acyl-CoA dehydrogenase (MCAD) and cholesterol oxidase (CHOX). In addition, the reduction potentials of lumiflavin in aqueous solution have also been computed. Using molecular dynamics and free-energy perturbation techniques, we obtained the free-energy changes for two-electron/two-proton as well as one-electron/one-proton addition steps. We employed a combined quantum mechanical and molecular mechanical (QM/MM) potential, in which the flavin ring was represented by the self-consistent-charge density functional tight-binding (SCC-DFTB) method, while the rest of the enzyme-solvent system was treated by classical force fields. The computed two-electron/two-proton reduction potentials for lumiflavin and the two enzyme-bound FADs are in reasonable agreement with experimental data. The calculations also yielded the pKa values for the one-electron reduced semiquinone (FH*) and the fully reduced hydroquinone (FH2) forms. The pKa of the FAD semiquinone in CHOX was found to be around 4, which is 4 units lower than that in the enzyme-free state and 2 units lower than that in MCAD; this supports the notion that oxidases have a greater ability than dehydrogenases to stabilize anionic semiquinones. In MCAD, the flavin ring interacts with four hydrophobic residues and has a significantly bent structure, even in the oxidized state. The present study shows that this bending of the flavin imparts a significant destabilization (approximately 5 kcal/mol) to the oxidized state. The reduction potential of lumiflavin was also computed using DFT (M06-L and B3LYP functionals with 6-31+G(d,p) basis set) with the SM6 continuum solvation model, and the results are in good agreement with results from explicit free-energy simulations, which supports the conclusion that the SCC-DFTB/MM computation is reasonably accurate for both 1e(-)/1H+ and 2e(-)/2H+ reduction processes. These results suggest that the first coupled electron-proton addition is stepwise for both the free and the two enzyme-bound flavins. In contrast, the second coupled electron-proton addition is also stepwise for the free flavin but is likely to be concerted when the flavin is bound to either the dehydrogenase or the oxidase enzyme.
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PMID:Combined quantum mechanical and molecular mechanical simulations of one- and two-electron reduction potentials of flavin cofactor in water, medium-chain acyl-CoA dehydrogenase, and cholesterol oxidase. 1756 13

The intramolecular and intermolecular perturbation on the electronic state of FAD was investigated by FTIR spectroscopy by using the C=O stretching vibrations as probes in D(2)O solution. Natural and artificial FADs, i.e. 8-CN-, 8-Cl-, 8-H-, 8-OCH(3)-, and 8-NH(2)-FAD labelled by 2-(13)C, (18)O=C(2), or 4,10a-(13)C(2) were used for band assignments. The C(2)=O and C(4)=O stretching vibrations of oxidized FAD were shifted systematically by the substitution at the 8-position, i.e. the stronger the electron-donating ability (NH(2) > OCH(3) > CH(3) > H > Cl > CN) of the substituent, the lower the wavenumber region where both the C(2)=O and C(4)=O bands appear. In contrast, the C(4)=O band of anionic reduced FAD scarcely shifted. The 1,645-cm(-1) band containing C(2)=O stretching vibration shifted to 1,630 cm(-1) in the medium-chain acyl-CoA dehydrogenase (MCAD)-bound state, which can be explained by hydrogen bonds at C(2)=O of the flavin ring. The band was observed at 1,607 cm(-1) in the complex of MCAD with 3-thiaoctanoyl-CoA. The 23 cm(-1) shift was explained by the charge-transfer interaction between oxidized flavin and the anionic acyl-CoA. In the case of electron-transferring flavoprotein, two bands associated with the C(4)=O stretching vibration were obtained at 1,712 and 1,686 cm(-1), providing evidence for the multiple conformations of the protein.
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PMID:Intramolecular and intermolecular perturbation on electronic state of FAD free in solution and bound to flavoproteins: FTIR spectroscopic study by using the C = O stretching vibrations as probes. 1787 56

Very-long-chain acyl-CoA dehydrogenase (VLCAD) is a member of the family of acyl-CoA dehydrogenases (ACADs). Unlike the other ACADs, which are soluble homotetramers, VLCAD is a homodimer associated with the mitochondrial membrane. VLCAD also possesses an additional 180 residues in the C terminus that are not present in the other ACADs. We have determined the crystal structure of VLCAD complexed with myristoyl-CoA, obtained by co-crystallization, to 1.91-A resolution. The overall fold of the N-terminal approximately 400 residues of VLCAD is similar to that of the soluble ACADs including medium-chain acyl-CoA dehydrogenase (MCAD). The novel C-terminal domain forms an alpha-helical bundle that is positioned perpendicular to the two N-terminal helical domains. The fatty acyl moiety of the bound substrate/product is deeply imbedded inside the protein; however, the adenosine pyrophosphate portion of the C14-CoA ligand is disordered because of partial hydrolysis of the thioester bond and high mobility of the CoA moiety. The location of Glu-422 with respect to the C2-C3 of the bound ligand and FAD confirms Glu-422 to be the catalytic base. In MCAD, Gln-95 and Glu-99 form the base of the substrate binding cavity. In VLCAD, these residues are glycines (Gly-175 and Gly-178), allowing the binding channel to extend for an additional 12A and permitting substrate acyl chain lengths as long as 24 carbons to bind. VLCAD deficiency is among the more common defects of mitochondrial beta-oxidation and, if left undiagnosed, can be fatal. This structure allows us to gain insight into how a variant VLCAD genotype results in a clinical phenotype.
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PMID:Structural basis for substrate fatty acyl chain specificity: crystal structure of human very-long-chain acyl-CoA dehydrogenase. 1822 65

Mitochondrial fatty acid oxidation defects have been recognized since the early 1970s. The discovery rate has been rather constant, with 3-4 'new' disorders identified every decade and with the most recent example, ACAD9 deficiency, reported in 2007. In this presentation we will focus on three of the 'old' defects: medium-chain acyl-CoA dehydrogenase (MCAD) deficiency, riboflavin responsive multiple acyl-CoA dehydrogenation (RR-MAD) deficiency, and short-chain acyl-CoA dehydrogenase (SCAD) deficiency. These disorders have been discussed in many publications and at countless conference presentations, and many questions relating to them have been answered. However, continuing clinical and pathophysiological research has raised many further questions, and new ideas and methodologies may be required to answer these. We will discuss these challenges. For MCAD deficiency the key question is why 80% of symptomatic patients are homozygous for the prevalent ACADM gene variation c.985A > G whereas this is found in only approximately 50% of newborns with a positive screen. For RR-MAD deficiency, the challenge is to find the connection between variations in the ETFDH gene and the observed deficiency of a number of different mitochondrial dehydrogenases as well as deficiency of FAD and coenzyme Q(10). With SCAD deficiency, the challenge is to elucidate whether ACADS gene variations are disease-associated, especially when combined with other genetic/cellular/environmental factors, which may act synergistically.
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PMID:Mitochondrial fatty acid oxidation defects--remaining challenges. 1883 89

The flavoenzyme nitroalkane oxidase is a member of the acyl-CoA dehydrogenase superfamily. Nitroalkane oxidase catalyzes the oxidation of neutral nitroalkanes to nitrite and the corresponding aldehydes or ketones. Crystal structures to 2.2 A resolution or better of enzyme complexes with bound substrates and of a trapped substrate-flavin adduct are described. The D402N enzyme has no detectable activity with neutral nitroalkanes [Valley, M. P., and Fitzpatrick, P. F. (2003) J. Am. Chem. Soc. 125, 8738-8739]. The structure of the D402N enzyme crystallized in the presence of 1-nitrohexane or 1-nitrooctane shows the presence of the substrate in the binding site. The aliphatic chain of the substrate extends into a tunnel leading to the enzyme surface. The oxygens of the substrate nitro group interact both with amino acid residues and with the 2'-hydroxyl of the FAD. When nitroalkane oxidase oxidizes nitroalkanes in the presence of cyanide, an electrophilic flavin imine intermediate can be trapped [Valley, M. P., Tichy, S. E., and Fitzpatrick, P. F. (2005) J. Am. Chem. Soc. 127, 2062-2066]. The structure of the enzyme trapped with cyanide during oxidation of 1-nitrohexane shows the presence of the modified flavin. A continuous hydrogen bond network connects the nitrogen of the CN-hexyl-FAD through the FAD 2'-hydroxyl to a chain of water molecules extending to the protein surface. Together, our complementary approaches provide strong evidence that the flavin cofactor is in the appropriate oxidation state and correlates well with the putative intermediate state observed within each of the crystal structures. Consequently, these results provide important structural descriptions of several steps along the nitroalkane oxidase reaction cycle.
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PMID:Crystal structures of intermediates in the nitroalkane oxidase reaction. 1926 37

The interactions of acyl-CoA with medium-chain acyl-CoA dehydrogenases (MCADs) reconstituted with artificial FADs-i.e. 8-CN-, 7,8-Cl(2)-, 8-Cl-, 8-OCH(3)- and 8-NH(2)-FAD-were investigated by UV-visible absorption and FT-IR measurements. Although 8-NH(2)-FAD-MCAD did not oxidize acyl-CoA the wavelength of the absorption maximum of the flavin was altered by acyl-CoAs binding. Thus, 8-NH(2)-FAD-MCAD is one of the attractive materials for investigation of enzyme-substrate (ES) interaction in ES complex (the complex of oxidized MCAD with acyl-CoA). FT-IR difference spectra between non-labelled and [1-(13)C]-labelled acyl-CoA free in solution and bound to oxidized 8-NH(2)-FAD-MCAD were obtained. The broad 1668-cm(-1) band of free octanoyl-CoA assigned to the C(1) = O stretching vibration appeared as a sharp signal at 1626 cm(-1) in the case of the complex. The downward shift indicates a large polarization of C(1) = O, and the sharpness suggests that the orientation of the C(1) = O in the active-site cavity is fairly limited. The hydrogen-bond enthalpy change responsible for the polarization on the transfer of the substrate from aqueous solution to the active site of MCAD was estimated to be approximately 15 kcal/mol. The 1626-cm(-1) band is noticeably weakened in the case of acyl-CoA with acyl chains longer than C12 which are poor substrates for MCAD, suggesting that C(1) = O is likely to exist in multiple orientations in the active-site cavity, whence the band becomes obscured. A band identical to that of bound C8-CoA was observed in the case of C4-CoA which is a poor substrate, indicating the strong hydrogen bond at C(1) = O.
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PMID:FT-IR spectroscopic studies on the molecular mechanism for substrate specificity/activation of medium-chain acyl-CoA dehydrogenase. 1947 May 21

Short-chain acyl-CoA dehydrogenase deficiency (SCADD) is an inborn error, biochemically characterized by increased plasma butyrylcarnitine (C4-C) concentration and increased ethylmalonic acid (EMA) excretion and caused by rare mutations and/or common gene variants in the SCAD encoding gene. Although its clinical relevance is not clear, SCADD is included in most US newborn screening programs. Riboflavin, the precursor of flavin adenine dinucleotide (FAD, cofactor), might be effective for treating SCADD. We assessed the FAD status and evaluated the effects of riboflavin treatment in a prospective open-label cohort study involving 16 patients with SCADD, subdivided into mutation/mutation (mut/mut), mutation/variant (mut/var), and variant/variant (var/var) genotype groups. Blood FAD levels were normal in all patients before therapy, but significantly lower in the mut/var and var/var groups compared with the mut/mut group. Riboflavin treatment resulted in a decrease in EMA excretion in the mut/var group and in a subjective clinical improvement in four patients from this group. However, this improvement persisted after stopping treatment. These results indicate that high-dose riboflavin treatment may improve the biochemical features of SCADD, at least in patients with a mut/var genotype and low FAD levels. As our study could not demonstrate a clinically relevant effect of riboflavin, general use of riboflavin cannot be recommended.
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PMID:Flavin adenine dinucleotide status and the effects of high-dose riboflavin treatment in short-chain acyl-CoA dehydrogenase deficiency. 1995 64

Anatoxin-a and homoanatoxin-a are two potent cyanobacterial neurotoxins. We recently reported the identification of the gene cluster responsible for the biosynthesis of these toxins in cyanobacteria and proposed a biosynthetic scheme starting from L-proline and involving three polyketide synthases for which the starter would be (S)-1-pyrroline-5-carboxylate bound to an acyl carrier protein, AnaD. We now report the in vitro reconstitution of the first steps of this biosynthesis in Oscillatoria PCC 6506. We identified in PCC 6506 the gene coding for an Sfp-like phosphopantetheinyl transferase and purified the gene product, OsPPT, that catalyzed the transfer of the phosphopantetheinyl arm to the serine 41 of AnaD. The pure adenylation protein AnaC loaded L-proline on holo-AnaD and was specific for L-proline (K(m) = 0.97 mM, k(cat) = 68 min(-1)) among the 20 natural amino acids. Among six close structural analogues of L-proline, including (S)-1-pyrroline-5-carboxylate, we only found 3,4-dehydro-L-proline to be an alternate substrate for AnaC (K(m) = 1.5 mM, k(cat) = 29 min(-1)). The putative prolyl-AnaD dehydrogenase, AnaB, purified to homogeneity as a histidine-tagged protein, showed an absorption spectrum characteristic of FAD-containing proteins. It oxidized prolyl-AnaD to dehydroprolyl-AnaD as shown by tryptic digestion of the protein followed by liquid chromatography coupled to tandem mass spectrometry. Alignment of the amino acid sequence of this dehydrogenase with related enzymes showed that AnaB belongs to the acyl-CoA dehydrogenase superfamily and thus probably catalyzes an alpha-beta-dehydrogenation of the thioester-bound proline followed by an aza-allylic isomerization to yield (S)-pyrroline-5-carboxyl-AnaD, the proposed starter for the subsequent polyketide synthase, AnaE.
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PMID:In vitro reconstitution of the first steps of anatoxin-a biosynthesis in Oscillatoria PCC 6506: from free L-proline to acyl carrier protein bound dehydroproline. 1995 30

D-kijanose is an unusual nitrosugar found attached to the antibiotic kijanimicin. Ten enzymes are required for its production in Actinomadura kijaniata, a soil-dwelling actinomycete. The focus of this investigation is on the protein encoded by the kijd3 gene and hereafter referred to as KijD3. On the basis of amino acid sequence analyses, KijD3 has been proposed to be an FAD-dependent oxidoreductase, which catalyzes the sixth step in d-kijanose biosynthesis by converting dTDP-3-amino-2,3,6-trideoxy-4-keto-3-methyl-d-glucose into its C-3' nitro derivative. This putative activity, however, has never been demonstrated in vivo or in vitro. Here we report the first structural study of this enzyme. For our investigation, crystals of KijD3 were grown in the presence of dTDP, and the structure was solved to 2.05-A resolution. The enzyme is a tetramer with each subunit folding into three distinct regions: a five alpha-helical bundle, an eight-stranded beta-sheet, and a second five alpha-helical bundle. The dTDP moiety is anchored to the protein via the side chains of Glu 113, Gln 254, and Arg 330. The overall fold of KijD3 places it into the well-characterized fatty acyl-CoA dehydrogenase superfamily. There is a decided cleft in each subunit with the appropriate dimensions to accommodate a dTDP-linked sugar. Strikingly, the loop defined by Phe 383 to Ala 388, which projects into the active site, contains two adjacent cis-peptide bonds, Pro 386 and Tyr 387. Activity assays demonstrate that KijD3 requires FAD for activity and that it produces a hydroxylamino product. The molecular architecture of KijD3 described in this report serves as a paradigm for a new family of enzymes that function on dTDP-linked sugar substrates.
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PMID:X-ray structure of kijd3, a key enzyme involved in the biosynthesis of D-kijanose. 2033 31

Electron transfer flavoprotein: ubiqionone oxidoreductase (ETF-QO) is a component of the mitochondrial respiratory chain that together with electron transfer flavoprotein (ETF) forms a short pathway that transfers electrons from 11 different mitochondrial flavoprotein dehydrogenases to the ubiquinone pool. The X-ray structure of the pig liver enzyme has been solved in the presence and absence of a bound ubiquinone. This structure reveals ETF-QO to be a monotopic membrane protein with the cofactors, FAD and a [4Fe-4S](+1+2) cluster, organised to suggests that it is the flavin that serves as the immediate reductant of ubiquinone. ETF-QO is very highly conserved in evolution and the recombinant enzyme from the bacterium Rhodobacter sphaeroides has allowed the mutational analysis of a number of residues that the structure suggested are involved in modulating the reduction potential of the cofactors. These experiments, together with the spectroscopic measurement of the distances between the cofactors in solution have confirmed the intramolecular pathway of electron transfer from ETF to ubiquinone. This approach can be extended as the R. sphaeroides ETF-QO provides a template for investigating the mechanistic consequences of single amino acid substitutions of conserved residues that are associated with a mild and late onset variant of the metabolic disease multiple acyl-CoA dehydrogenase deficiency (MADD).
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PMID:The electron transfer flavoprotein: ubiquinone oxidoreductases. 2093 44


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