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)

Acyl-CoA dehydrogenases constitute a family of flavoproteins that catalyze the alpha,beta-dehydrogenation of fatty acid acyl-CoA conjugates. While they differ widely in their specificity, they share the same basic chemical mechanism of alpha,beta-dehydrogenation. Medium chain acyl-CoA dehydrogenase is probably the best-studied member of the class and serves as a model for the study of catalytic mechanisms. Based on medium chain acyl-CoA dehydrogenase we discuss the main factors that bring about catalysis, promote specificity and determine the selective transfer of electrons to electron transferring flavoprotein. The mechanism of alpha,beta-dehydrogenation is viewed as a process in which the substrate alphaC-H and betaC-H bonds are ruptured concertedly, the first hydrogen being removed by the active center base Glu376-COO- as an H+, the second being transferred as a hydride to the flavin N(5) position. Hereby the pKa of the substrate alphaC-H is lowered from > 20 to approximately 8 by the effect of specific hydrogen bonds. Concomitantly, the pKa of Glu376-COO- is also raised to 8-9 due to the decrease in polarity brought about by substrate binding. The kinetic sequence of medium chain acyl-CoA dehydrogenase is rather complex and involves several intermediates. A prominent one is the molecular complex of reduced enzyme with the enoyl-CoA product that is characterized by an intense charge transfer absorption and serves as the point of transfer of electrons to the electron transferring flavoprotein. These views are also discussed in the context of the accompanying paper on the three-dimensional properties of acyl-CoA dehydrogenases.
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PMID:Acyl-CoA dehydrogenases. A mechanistic overview. 1472 76

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

A gene (PP2216) that codes for an acyl-CoA dehydrogenase was cloned from Pseudomonas putida strain KT2240 and over-expressed in Escherichia coli, and the recombinant enzyme purified and characterised. The enzyme is tetrameric with one FAD per subunit of molecular mass 40,500 Da. An anaerobic titration with sodium dithionite showed that the enzyme accepts two electrons. A similar titration with butyryl-CoA showed that reduction by this substrate was incomplete with 4.5 mol butyryl-CoA added per mol enzyme FAD; the equilibrium was used to calculate that the oxidation-reduction potential of the enzyme at pH 7 and 25 degrees C is 5+/-5 mV versus the standard hydrogen electrode. The enzyme shows catalytic activity with butyryl-CoA, valeryl-CoA and hexanoyl-CoA, and very low activity with heptanoyl-CoA and octanoyl-CoA; it fails to oxidise propionyl-CoA. These properties resemble those of short-chain acyl-CoA dehydrogenases from other sources. The enzyme is inactive with the CoA derivatives of all phenylalkanoates that were tested (side chains 3-8 carbon atoms) indicating that in contrast to an earlier suggestion, the enzyme is not involved in the beta-oxidation of aromatic compounds.
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PMID:The protein coded by the PP2216 gene of Pseudomonas putida KT2440 is an acyl-CoA dehydrogenase that oxidises only short-chain aliphatic substrates. 1602 85

Potential of mean force calculations have been performed on the wild-type medium-chain acyl-CoA dehydrogenase (MCAD) and two of its mutant forms. Initial simulation and analysis of the active site of the enzyme reveal that an arginine residue (Arg256), conserved in the substrate-binding domain of this group of enzymes, exists in two alternate conformations, only one of which makes the enzyme active. This active conformation was used in subsequent computations of the enzymatic reactions. It is known that the catalytic alpha,beta-dehydrogenation of fatty acyl-CoAs consists of two C-H bond dissociation processes: a proton abstraction and a hydride transfer. Energy profiles of the two reaction steps in the wild-type MCAD demonstrate that the reaction proceeds by a stepwise mechanism with a transient species. The activation barriers of the two steps differ by only approximately 2 kcal/mol, indicating that both may contribute to the rate-limiting process. Thus this may be a stepwise dissociation mechanism whose relative barriers can be tuned by suitable alterations of the substrate and/or enzyme. Analysis of the structures along the reaction path reveals that Arg256 plays a key role in maintaining the reaction center hydrogen-bonding network involving the thioester carbonyl group, which stabilizes transition states as well as the intervening transient species. Mutation of this arginine residue to glutamine increases the activation barrier of the hydride transfer reaction by approximately 5 kcal/mol, and the present simulations predict a substantial loss of catalytic activity for this mutant. Structural analysis of this mutant reveals that the orientation of the thioester moiety of the substrate has been changed significantly as compared to that in the wild-type enzyme. In contrast, simulation of the active site of the Thr168Ala mutant shows no significant change in the relative orientation of the substrate and the cofactor in the active site; as a result, this mutation has very little effect on the overall reaction barrier, and this is consistent with the experimental data. This study demonstrates that significant insights into the catalytic mechanism can be obtained from simulation studies, and the results can be used to design novel mechanistic probes for the enzyme.
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PMID:Potential of mean force calculation for the proton and hydride transfer reactions catalyzed by medium-chain acyl-CoA dehydrogenase: effect of mutations on enzyme catalysis. 1634 46

Nitroalkane oxidase (NAO) from Fusarium oxysporum catalyzes the oxidation of neutral nitroalkanes to the corresponding aldehydes or ketones with the production of H(2)O(2) and nitrite. The flavoenzyme is a new member of the acyl-CoA dehydrogenase (ACAD) family, but it does not react with acyl-CoA substrates. We present the 2.2 A resolution crystal structure of NAO trapped during the turnover of nitroethane as a covalent N5-FAD adduct (ES*). The homotetrameric structure of ES* was solved by MAD phasing with 52 Se-Met sites in an orthorhombic space group. The electron density for the N5-(2-nitrobutyl)-1,5-dihydro-FAD covalent intermediate is clearly resolved. The structure of ES was used to solve the crystal structure of oxidized NAO at 2.07 A resolution. The c axis for the trigonal space group of oxidized NAO is 485 A, and there are six subunits (1(1)/(2) holoenzymes) in the asymmetric unit. Four of the active sites contain spermine (EI), a weak competitive inhibitor, and two do not contain spermine (E(ox)). The active-site structures of E(ox), EI, and ES* reveal a hydrophobic channel that extends from the exterior of the protein and terminates at Asp402 and the N5 position on the re face of the FAD. Thus, Asp402 is in the correct position to serve as the active-site base, where it is proposed to abstract the alpha proton from neutral nitroalkane substrates. The structures for NAO and various members of the ACAD family overlay with root-mean-square deviations between 1.7 and 3.1 A. The homologous region typically spans more than 325 residues and includes Glu376, which is the active-site base in the prototypical member of the ACAD family. However, NAO and the ACADs exhibit differences in hydrogen-bonding patterns between the respective active-site base, substrate molecules, and FAD. These likely differentiate NAO from the homologues and, consequently, are proposed to result in the unique reaction mechanism of NAO.
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PMID:Crystal structures of nitroalkane oxidase: insights into the reaction mechanism from a covalent complex of the flavoenzyme trapped during turnover. 1643 Feb 10

The three-dimensional structure of rat-liver acyl-CoA oxidase-II (ACO-II) in a complex with a C12-fatty acid was solved by the molecular replacement method based on the uncomplexed ACO-II structure. The crystalline form of the complex was obtained by cocrystallization of ACO-II with dodecanoyl-CoA. The crystalline complex possessed, in the active-site crevice, only the fatty acid moiety that had been formed through hydrolysis of the thioester bond. The overall dimeric structure and the folding pattern of each subunit are essentially superimposable on those of uncomplexed ACO-II. The active site including the flavin ring of FAD, the crevice embracing the fatty acyl moiety, and adjacent amino acid side chains are superimposably conserved with the exception of Glu421, whose carboxylate group is tilted away to accommodate the fatty acid. One of the carboxyl oxygens of the bound fatty acid is hydrogen-bonded to the amide hydrogen of Glu421, the presumed catalytic base, and to the ribityl 2'-hydroxyl group of FAD. This hydrogen-bonding network correlates well with the substrate recognition/activation in acyl-CoA dehydrogenase. The binding mode of C12-fatty acid suggests that the active site does not close upon substrate binding, but remains spacious during the entire catalytic process, the oxygen accessibility in the oxidative half-reaction thereby being maintained.
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PMID:Three-dimensional structure of rat-liver acyl-CoA oxidase in complex with a fatty acid: insights into substrate-recognition and reactivity toward molecular oxygen. 1667 80

p-Hydroxyphenylacetate hydroxylase from Acinetobacter baumannii is a two-component system consisting of a NADH-dependent FMN reductase and a monooxygenase (C2) that uses reduced FMN as substrate. The crystal structures of C2 in the ligand-free and substrate-bound forms reveal a preorganized pocket that binds reduced FMN without large conformational changes. The Phe-266 side chain swings out to provide the space for binding p-hydroxyphenylacetate that is oriented orthogonal to the flavin ring. The geometry of the substrate-binding site of C2 is significantly different from that of p-hydroxybenzoate hydroxylase, a single-component flavoenzyme that catalyzes a similar reaction. The C2 overall structure resembles the folding of medium-chain acyl-CoA dehydrogenase. An outstanding feature in the C2 structure is a cavity located in front of reduced FMN; it has a spherical shape with a 1.9-A radius and a 29-A3 volume and is interposed between the flavin C4a atom and the substrate atom to be hydroxylated. The shape and position of this cavity are perfectly fit for housing the oxygen atoms of the flavin C4a-hydroperoxide intermediate that is formed upon reaction of the C2-bound reduced flavin with molecular oxygen. The side chain of His-396 is predicted to act as a hydrogen-bond donor to the oxygen atoms of the intermediate. This architecture promotes the nucleophilic attack of the substrate onto the terminal oxygen of the hydroperoxyflavin. Comparative analysis with the structures of other flavoenzymes indicates that a distinctive feature of monooxygenases is the presence of specific cavities that encapsulate and stabilize the crucial hydroperoxyflavin intermediate.
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PMID:Structure of the monooxygenase component of a two-component flavoprotein monooxygenase. 1722 49

Glu376, the base involved in substrate alphaH+ abstraction at the active center of medium-chain acyl-CoA dehydrogenase (MCAD), has been mutated to Gln and Gly. The mutants are active; however, their rates of dehydrogenation are lowered by approximately 5 orders of magnitude. Binding of the substrate octanoyl-CoA to Glu376Gln-MCAD involves (at least) two steps. The ensuing dehydrogenation reaction that corresponds to reduction of the flavin cofactor also occurs in two phases. These are interpreted to consist of a first, reversible step, followed by a slower, practically irreversible one. For Glu376Gln-MCAD, the log of the rates of dehydrogenation increases linearly with pH (slope = 1) in the pH range of 6-10, suggesting HO- as a reactant. The rates of the same reactions in D2O have the same pD profile and reflect a solvent kinetic isotope effect (SKIE) of approximately 8.5. Glu376Gln+Glu99Gly-MCAD (studied to assess the role of Glu99 also present at the bottom of the active center cavity) has activities and activity profiles similar to those of Glu376Gln-MCAD. This excludes Glu99 as the active center base for Glu376Gln-MCAD catalysis. Proton inventories for the two phases of the dehydrogenation reaction were investigated at 4 and 25 degrees C. The inventories at 25 degrees C reflect a SKIE of approximately 4.5; the profiles are "bowl-shaped", in which a transition-state contribution predominates. The profiles for the 4 degrees C reaction are very unusual. That for the first phase can be analyzed on a two-step model with one step (80% rate-limiting) having a conformational reorganization with an isotope effect of 90-100, from small isotope effects at many protein sites, and the other step (20% rate-limiting) having an inverse isotope effect of ca. 2, characteristic of the reaction of hydroxide ion as a base. For the second phase, only a contribution from many protein sites with a KIE of approximately 4.5 is observed. The results are compatible with a very rigid active site framework that must undergo rearrangements for dehydrogenation to take place, and specifically to allow access of HO-, the reactant that must neutralize the H+ abstracted from the alphaC-H substrate. The large isotope effects are attributed to the changes in state of several H-bonds that occur during the process.
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PMID:Solvent isotope effects in reactions of human medium-chain acyl-CoA dehydrogenase active site mutants. 1728 88

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

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


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