Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: KEGG:D02011 (FAD)
5,530 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The crystal structure of electron transfer flavoprotein (ETF) from Paracoccus denitrificans was determined and refined to an R-factor of 19.3% at 2.6 A resolution. The overall fold is identical to that of the human enzyme, with the exception of a single loop region. Like the human structure, the structure of the P. denitrificans ETF is comprised of three distinct domains, two contributed by the alpha-subunit and the third from the beta-subunit. Close analysis of the structure reveals that the loop containing betaI63 is in part responsible for conferring the high specificity of AMP binding by the ETF protein. Using the sequence and structures of the human and P. denitrificans enzymes as models, a detailed sequence alignment has been constructed for several members of the ETF family, including sequences derived for the putative FixA and FixB proteins. From this alignment, it is evident that in all members of the ETF family the residues located in the immediate vicinity of the FAD cofactor are identical, with the exception of the substitution of serine and leucine residues in the W3A1 ETF protein for the human residues alphaT266 and betaY16, respectively. Mapping of ionic differences between the human and P. denitrificans ETF onto the structure identifies a surface that is electrostatically very similar between the two proteins, thus supporting a previous docking model between human ETF and pig medium-chain acyl-CoA dehydrogenase (MCAD). Analysis of the ionic strength dependence of the electron transfer reaction between either human or P. denitrificans ETF and MCAD demonstrates that the human ETF functions optimally at low ( approximately 10 mequiv) ionic strength, while P. denitrificans ETF is a better electron acceptor at higher (>75 mequiv) ionic strength. This suggests that the electrostatic surface potential of the two proteins is very different and is consistent with the difference in isoelectric points between the proteins. Analysis of the electrostatic potentials of the human and P. denitrificans ETFs reveals that the P. denitrificans ETF is more negatively charged. This excess negative charge may contribute to the difference in redox potentials between the two ETF flavoproteins and suggests an explanation for the opposing ionic strength dependencies for the reaction of MCAD with the two ETFs. Furthermore, by analysis of a model of the previously described human-P. denitrificans chimeric ETF protein, it is possible to identify one region of ETF that participates in docking with ETF-ubiquinone oxidoreductase, the physiological electron acceptor for ETF.
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PMID:Crystal structure of Paracoccus denitrificans electron transfer flavoprotein: structural and electrostatic analysis of a conserved flavin binding domain. 1002 81

The redox-inactive thioester analog 3-thia-octanoyl-CoA blocks transfer of a hydride equivalent to the flavin prosthetic group of the medium-chain acyl-CoA dehydrogenase with the accumulation of a stable enolate intermediate not encountered with normal substrates. Substitution of the normal flavin with 5-deaza-FAD would thus be expected to lead to enolate formation with both normal and 3-thia-substrate analogs, because reduction of the 5-deaza-enzyme is thermodynamically highly unfavorable. However, spectrophotometric titrations show that neither ligand forms significant enolate species with the 5-deaza-FAD enzyme. Similarly, the substituted dehydrogenase catalyzes undetectable alpha-proton exchange with octanoyl-CoA and ca. 1% of the corresponding rate with 3-thia-octanoyl-CoA when compared to the native enzyme. This inability to stabilize enolate species is not simply due to impaired binding of CoA-thioester analogs, because binding of a range of ligands is weakened by only 2- to 10-fold with the 5-deaza-enzyme. 4-Thia-trans-2-enoyl-CoA product is polarized normally on binding to the substituted protein, showing that this critical aspect of catalysis is apparently normal. These data, together with studies with CoA-persulfide and acetoacetyl- and p-nitrophenylacetyl-CoA, suggest that 5-deaza-FAD substitution exerts subtle, unanticipated, effects on the reductive half-reaction of the medium-chain acyl-CoA dehydrogenase. The involvement of charge-transfer interactions in the acidification of weakly acidic acyl-CoA thioesters is discussed.
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PMID:Thioester enolate stabilization in the acyl-CoA dehydrogenases: the effect of 5-deaza-flavin substitution. 1148 11

The flavoprotein nitroalkane oxidase (NAO) from Fusarium oxysporum catalyzes the oxidation of nitroalkanes to the respective aldehydes with production of nitrite and hydrogen peroxide. The sequences of several peptides from the fungal enzyme were used to design oligonucleotides for the isolation of a portion of the NAO gene from an F. oxysporum genomic DNA preparation. This sequence was used to clone the cDNA for NAO from an F. oxysporum cDNA library. The sequence of the cloned cDNA showed that NOA is a member of the acyl-CoA dehydrogenase (ACAD) superfamily. The members of this family share with NAO a mechanism that is initiated by proton removal from carbon, suggesting a common chemical reaction for this superfamily. NAO was expressed in Escherichia coli and the recombinant enzyme was characterized. Recombinant NAO has identical kinetic parameters to enzyme isolated from F. oxysporum but is isolated with oxidized FAD rather than the nitrobutyl-FAD found in the fungal enzyme. NAO purified from E. coli or from F. oxysporum has no detectable ACAD activity on short- or medium-chain acyl CoAs, and medium-chain acyl-CoA dehydrogenase and short-chain acyl-CoA dehydrogenase are unable to catalyze oxidation of nitroalkanes.
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PMID:Cloning of nitroalkane oxidase from Fusarium oxysporum identifies a new member of the acyl-CoA dehydrogenase superfamily. 1186 31

Acyl-CoA oxidase (ACO) catalyzes the first and rate-determining step of the peroxisomal beta-oxidation of fatty acids. The crystal structure of ACO-II, which is one of two forms of rat liver ACO (ACO-I and ACO-II), has been solved and refined to an R-factor of 20.6% at 2.2-A resolution. The enzyme is a homodimer, and the polypeptide chain of the subunit is folded into the N-terminal alpha-domain, beta-domain, and C-terminal alpha-domain. The X-ray analysis showed that the overall folding of ACO-II less C-terminal 221 residues is similar to that of medium-chain acyl-CoA dehydrogenase (MCAD). However, the N-terminal alpha- and beta-domains rotate by 13 with respect to the C-terminal alpha-domain compared with those in MCAD to give a long and large crevice that accommodates the cofactor FAD and the substrate acyl-CoA. FAD is bound to the crevice between the beta- and C-terminal domains with its adenosine diphosphate portion interacting extensively with the other subunit of the molecule. The flavin ring of FAD resides at the active site with its si-face attached to the beta-domain, and is surrounded by active-site residues in a mode similar to that found in MCAD. However, the residues have weak interactions with the flavin ring due to the loss of some of the important hydrogen bonds with the flavin ring found in MCAD. The catalytic residue Glu421 in the C-terminal alpha-domain seems to be too far away from the flavin ring to abstract the alpha-proton of the substrate acyl-CoA, suggesting that the C-terminal domain moves to close the active site upon substrate binding. The pyrimidine moiety of flavin is exposed to the solvent and can readily be attacked by molecular oxygen, while that in MCAD is protected from the solvent. The crevice for binding the fatty acyl chain is 28 A long and 6 A wide, large enough to accommodate the C23 acyl chain.
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PMID:Three-dimensional structure of the flavoenzyme acyl-CoA oxidase-II from rat liver, the peroxisomal counterpart of mitochondrial acyl-CoA dehydrogenase. 1187 65

The active site residue, Glu-376, of medium-chain acyl-CoA dehydrogenase (MCAD) has been known to abstract the alpha-proton from acyl-CoA substrates during the course of the reductive half-reaction. The site-specific mutation of Glu-376-->Gln(E376Q) slows down the octanoyl-CoA-dependent reductive half-reaction of the enzyme by about 5 orders of magnitude due to impairment in the proton-transfer step. To test whether the carboxyl group of Glu-376 exclusively serves as the active site base (for abstracting the alpha-proton) during the enzyme catalysis, we undertook a detailed kinetic investigation of the enzyme-ligand interaction and enzyme catalysis, utilizing octanoyl-CoA/octenoyl-CoA as a physiological substrate/product pair and the wild-type and E376Q mutant enzymes as the catalysts. The transient kinetic data revealed that the E376Q mutation not only impaired the rate of octanoyl-CoA-dependent reduction of the enzyme-bound FAD, but also impaired the association and dissociation rates for the binding of the reaction product, octenoyl-CoA. Besides, the E376Q mutation correspondingly impaired the kinetic profiles for the quenching of the intrinsic protein fluorescence during the course of the above diverse (i.e., "chemistry" versus "physical interaction") processes. A cumulative account of the experimental data led to the suggestion that the carboxyl group of Glu-376 of MCAD is intimately involved in modulating the microscopic environment (protein conformation) of the enzyme's active site during the course of ligand binding and catalysis. Arguments are presented that the electrostatic interactions among Glu-376, FAD, and CoA-ligands are responsible for structuring the enzyme's active site cavity in the ground and transition states of the enzyme during the above physicochemical processes.
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PMID:Beyond the proton abstracting role of Glu-376 in medium-chain acyl-CoA dehydrogenase: influence of Glu-376-->Gln substitution on ligand binding and catalysis. 1192 26

The role of the oxyanion hole in the reaction catalyzed by pig medium-chain acyl-CoA dehydrogenase (pMCAD) has been investigated using enzyme reconstituted with 2'-deoxy-FAD. The k(cat) (18.8 +/- 0.5 s(-1)) and K(m) (2.5 +/- 0.4 microM) values for the oxidation of n-octanoyl-CoA (C(8)-CoA) by WT pMCAD recombinantly expressed in Escherichia coli are similar to those of native pMCAD isolated from pig kidney. In agreement with previous studies [Engst et al. (1999) Biochemistry 38, 257-267], reconstitution of the WT enzyme with 2'-deoxy-FAD causes a large (400-fold) decrease in k(cat) but has little effect on K(m). To investigate the molecular basis for the alterations in activity resulting from changes in hydrogen bonding between the substrate and the enzyme's oxyanion hole, the structure of the product analogue hexadienoyl-CoA (HD-CoA) bound to the 2'-deoxy-FAD-reconstituted enzyme has been probed by Raman spectroscopy. Importantly, while WT pMCAD causes a 27 cm(-1) decrease in the vibrational frequency of the HD enone band, from 1595 to 1568 cm(-1), the enone band is only shifted 10 cm(-1) upon binding HD-CoA to 2'-deoxy-FAD pMCAD. Thus, removal of the 2'-ribityl hydroxyl group results in a substantial reduction in the ability of the enzyme to polarize the ground state of the ES complex. On the basis of an analysis of a similar system, it is estimated that ground state destabilization is reduced by up to 17 kJ mol(-1), while the activation energy for the reaction is raised 15 kJ mol(-1). In addition, removal of the 2'-ribityl hydroxyl reduces the redox potential shift that is induced by HD-CoA binding from 18 to 11 kJ mol(-1). Consequently, while ligand polarization caused by hydrogen bonding in the oxyanion hole is intimately linked to substrate turnover, additional factors must be responsible for ligand-induced changes in redox potential. Finally, while replacement of the catalytic base E376 with Gln abolishes the ability of the enzyme to catalyze substrate oxidation and to catalyze the exchange of the C(8)-CoA alpha-protons with solvent deuterium, the 2'-deoxy-FAD-reconstituted enzyme catalyzes alpha-proton exchange at a rate (k(exc)) of 0.085 s(-1), which is only 4-fold slower than k(exc) for WT pMCAD (0.35 s(-1)). Thus, either the oxyanion hole plays only a minor role in stabilizing the transition state for alpha-proton exchange, in contrast to its role in substrate oxidation, or the value of k(exc) for WT pMCAD reflects a process such as exchange of the E376 COOH proton with solvent.
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PMID:Probing hydrogen-bonding interactions in the active site of medium-chain acyl-CoA dehydrogenase using Raman spectroscopy. 1452 97

The pKa value of a substrate analogue 3-thiaoctanoyl-CoA at alphaC-H is known to drop from ca. 16 in the free state to 5-6 upon binding to medium-chain acyl-CoA dehydrogenase (MCAD). The molecular mechanism underlying this phenomenon was investigated by taking advantage of artificial FADs, i.e., 8-CN-, 7,8-Cl2-, 8-Cl-, 8-OCH3-, 8-NH2-, ribityl-2'-deoxy-8-CN-, and ribityl-2'-deoxy-8-Cl-FADs, reconstituted into MCAD. The stronger the electron-withdrawing ability of the substituent, the smaller the pKa value became [e.g., 7.4 (8-NH2-FAD) and 4.0 (8-CN-FAD)], suggesting that the flavin ring itself affects the pKa value of the ligand via a charge-transfer interaction with the ligand. The destruction of the hydrogen bond between the thioester C(1)=O and the ribityl-2'-OH of FAD raised the pKa by ca. 2.5 units. These results indicate that the interaction between the ligand and the flavin ring also serves to lower the pKa of the ligand, in addition to the hydrogen bonds at C(1)=O of the ligand.
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PMID:Molecular mechanism of the drop in the pKa of a substrate analog bound to medium-chain acyl-CoA dehydrogenase: implications for substrate activation. 1476 72

Mitochondrial medium-chain acyl-CoA dehydrogenase is a key enzyme for the beta oxidation of fatty acids, and the deficiency of this enzyme in patients has been previously reported. We found that the enzyme has intrinsic isomerase activity, which was confirmed using incubation followed with HPLC analysis. The isomerase activity of the enzyme was thoroughly characterized through studies of kinetics, substrate specificity, pH dependence, and enzyme inhibition. E376 mutants were constructed, and mutant enzymes were purified and characterized. It was shown that E376 is the catalytic residue for both dehydrogenase and isomerase activities of the enzyme. The isomerase activity of medium-chain acyl-CoA dehydrogenase is probably a spontaneous process driven by thermodynamic equilibrium with the formation of a conjugated structure after deprotonation of substrate alpha proton. The energy level of the transition state may be lowered by a stable dienolate intermediate, which gains further stabilization via charge transfer with the electron-deficient FAD cofactor of the enzyme. This raises the question as to whether the dehydrogenase might function as an isomerase in vivo in conditions in which the activity of the isomerase is decreased.
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PMID:Intrinsic isomerase activity of medium-chain acyl-CoA dehydrogenase. 1585 Apr 6

Mitochondrial medium-chain acyl-CoA dehydrogenase is a key enzyme for the beta-oxidation of fatty acids, which catalyzes the FAD-dependent oxidation of a variety of acyl-CoA substrates to the corresponding trans-2-enoyl-CoA thioesters. Oct-4-en-2-ynoyl-CoA was identified as a new irreversible inhibitor of acyl-CoA dehydrogenase, and kinetic parameters K(I) and k(inact) were determined to be 11 microM and 0.025 min(-1), respectively. Triple bond between C2 and C3 of the inhibitor was identified as the functional group responsible for enzyme inactivation, and Michael addition is proposed as the mechanism for this inactivation, which is a new pathway for inactivation of MCAD by inhibitors. The inhibitor may become a lead for further development for treating non-insulin-dependent diabetes mellitus.
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PMID:Inactivation of medium-chain acyl-CoA dehydrogenase by oct-4-en-2-ynoyl-CoA. 1629 16

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


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