Mechanistic and structural analyses of the roles of Arg409 and Asp402 in the reaction of the flavoprotein nitroalkane oxidase

The flavoprotein nitroalkane oxidase (NAO) catalyzes the oxidation of primary and secondary nitroalkanes to the corresponding aldehydes and ketones. The enzyme is a homologue of acyl-CoA dehydrogenase. Asp402 in NAO has been proposed to be the active site base responsible for removing the substrate proton in the first catalytic step; structurally it corresponds to the glutamate which acts as the base in medium chain acyl-CoA dehydrogenase. In the active site of NAO, the carboxylate of Asp402 forms an ionic interaction with the side chain of Arg409. The R409K enzyme has now been characterized kinetically and structurally. The mutation results in a decrease in the rate constant for proton abstraction of 100-fold. Analysis of the three-dimensional structure of the R409K enzyme, determined by X-ray crystallography to a resolution of 2.65 A, shows that the critical structural change is an increase in the distance between the carboxylate of Asp402 and the positively charged nitrogen in the side chain of the residue at position 409. The D402E mutation results in a smaller decrease in the rate constant for proton abstraction of 18-fold. The structure of the D402E enzyme, determined at 2.4 A resolution, shows that there is a smaller increase in the distance between Arg409 and the carboxylate at position 402, and the interaction of this residue with Ser276 is perturbed. These results establish the critical importance of the interaction between Asp402 and Arg409 for proton abstraction by nitroalkane oxidase.     

Reductive half-reaction of nitroalkane oxidase: effect of mutation of the active site aspartate to glutamate

The flavoenzyme nitroalkane oxidase catalyzes the oxidation of primary and secondary nitroalkanes to the respective aldehydes or ketones, releasing nitrite. The enzyme has recently been identified as being homologous to the acyl-CoA dehydrogenase family of enzymes [Daubner, S. C., Gadda, G., Valley, M. P., and Fitzpatrick, P. F. (2002) Proc. Natl. Acad. Sci. U.S.A. 99, 2702-2707]. The glutamate which acts as an active site base in that family of enzymes aligns with Asp402 of nitroalkane oxidase. To evaluate the identification of Asp402 as an active site base, the effect of mutation of Asp402 to glutamate on the rate of cleavage of the nitroalkane C-H bond has been determined. Deuterium kinetic isotope effects on steady state kinetic parameters and direct measurement of the rate of flavin reduction establish that the mutation increases the DeltaG(++) for C-H bond cleavage by 1.6-1.9 kcal/mol. There is no effect on the rate of reaction of the reduced enzyme with oxygen. These results support the assignment of Asp402 as the active site base in nitroalkane oxidase.     

Cloning and characterization of a human cDNA ACAD10 mapped to chromosome 12q24.1

Mitochondrial fatty acid -oxidation is an important energy resource for many mammal tissues. Acyl-CoA dehydrogenases (ACADs) are a family of flavoproteins that are involved in the -oxidation of the fatty acyl-CoA derivatives. Deficiency of these ACADs can cause metabolic disorders including muscle fatigue, hypoglycaemia, hepatic lipidosis and so on. By large scale sequencing, we identified a cDNA sequence of 3960 base pairs with a typical acyl-CoA dehydrogenase function domain. RT-PCR result shows that it is widely expressed in human tissues, especially high in liver, kidney, pancreas and spleen. It is hypothesized that this is a novel member of ACADs family. Abbreviations: ACADs – acyl-CoA dehydrogenases, FAD – flavinadenine dinucleotide, SCAD – short-chain acyl-CoA dehydrogenase,MCAD – medium-chain acyl-CoA dehydrogenase, LCAD – long-chain acyl-CoAdehydrogenase, VLCAD – very long- chain acyl-CoA dehydrogenase, IVD –isocalery-CoA dehydrogenase, SBCAD – short/branched chain acyl-CoAdehydrogenase, GCD – glutaryl- CoA dehydrogenase, ETF – electron transferflavoprotein, ACAD8 – acyl-CoA dehydrogenase 8, ACAD9 – acyl-CoAdehydrogenase 9, ACAD10 – acyl-CoA dehydrogenase 10.     

Impact of the N5-proximal Asn on the thermodynamic and kinetic stability of the semiquinone radical in photolyase

Flavoproteins can dramatically adjust the thermodynamics and kinetics of electron transfer at their flavin cofactor. A versatile regulatory tool is proton transfer. Here, we demonstrate the significance of proton-coupled electron transfer to redox tuning and semiquinone (sq) stability in photolyases (PLs) and cryptochromes (CRYs). These light-responsive proteins share homologous overall architectures and FAD-binding pockets, yet they have evolved divergent functions that include DNA repair, photomorphogenesis, regulation of circadian rhythm, and magnetoreception. We report the first measurement of both FAD redox potentials for cyclobutane pyrimidine dimer PL (CPD-PL, Anacystis nidulans). These values, E(1)(hq/sq) = -140 mV and E(2)(sq/ox) = -219 mV, where hq is FAD hydroquinone and ox is oxidized FAD, establish that the sq is not thermodynamically stabilized (ΔE = E(2) - E(1) = -79 mV). Results with N386D CPD-PL support our earlier hypothesis of a kinetic barrier to sq oxidation associated with proton transfer. Both E(1) and E(2) are upshifted by ～ 100 mV in this mutant; replacing the N5-proximal Asn with Asp decreases the driving force for sq oxidation. However, this Asp alleviates the kinetic barrier, presumably by acting as a proton shuttle, because the sq in N386D CPD-PL oxidizes orders of magnitude more rapidly than wild type. These data clearly reveal, as suggested for plant CRYs, that an N5-proximal Asp can switch on proton transfer and modulate sq reactivity. However, the effect is context-dependent. More generally, we propose that PLs and CRYs tune the properties of their N5-proximal residue to adjust the extent of proton transfer, H-bonding patterns, and changes in protein conformation associated with electron transfer at the flavin.     

Yeast Fms1 is a FAD-utilizing polyamine oxidase

In this report we show that recombinant Saccharomyces cerevisiae Fms1 protein is a polyamine oxidase that binds FAD with an FAD:Fms1 stoichiometry of 1:1. Biochemical characterization of Fms1 shows that it can oxidize spermine, N(1)-acetylspermine, N(1)-acetylspermidine, and N(8)-acetylspermidine, but not spermidine. The products of spermine oxidation are spermidine and 3-aminopropanal. A kinetic analysis revealed that spermine, N(1)-acetylspermine, and N(1)-acetylspermidine are oxidized with similar efficiencies, while N(8)-acetylspermidine is a poor substrate. The data support a previous report, suggesting that Fms1 is responsible for the production of beta-alanine from spermine for the synthesis of pantothenic acid.     

A fluorescently labeled undecapeptide derived from a protein in royal jelly of the honeybee—royalisin—for specific detection of oxidized low‐density lipoprotein

The probes for detection of oxidized low‐density lipoprotein (ox‐LDL) in plasma and in atherosclerotic plaques are expected to facilitate the diagnosis, prevention, and treatment of atherosclerosis. Recently, we have reported that a heptapeptide (Lys‐Trp‐Tyr‐Lys‐Asp‐Gly‐Asp, KP6) coupled through the ε‐amino group of N‐terminal Lys to fluorescein isothiocyanate (FITC), (FITC)KP6, can be useful as a fluorescent probe for specific detection of ox‐LDL. In the present study, to develop a novel fluorescent peptide for specific detection of ox‐LDL, we investigated the interaction (with ox‐LDL) of an undecapeptide corresponding to positions 41 to 51 of a potent antimicrobial protein (royalisin, which consists of 51 residues; from royal jelly of honeybees), conjugated at the N‐terminus to FITC in the presence of 6‐amino‐n‐caproic acid (AC) linker, (FITC‐AC)‐royalisin P11, which contains both sequences, Phe‐Lys‐Asp and Asp‐Lys‐Tyr, similar to Tyr‐Lys‐Asp in (FITC)KP6. The (FITC‐AC)‐royalisin P11 bound with high specificity to ox‐LDL in a dose‐dependent manner, through the binding to major lipid components in ox‐LDL (lysophosphatidylcholine and oxidized phosphatidylcholine). In contrast, a (FITC‐AC)‐shuffled royalisin P11 peptide, in which sequences Phe‐Lys‐Asp and Asp‐Lys‐Tyr were modified to Lys‐Phe‐Asp and Asp‐Tyr‐Lys, respectively, hardly bound to LDL and ox‐LDL. These findings strongly suggest that (FITC‐AC)‐royalisin P11 may be an effective fluorescent probe for specific detection of ox‐LDL and that royalisin from the royal jelly of honeybees may play a role in the treatment of atherosclerosis through the specific binding of the region at positions 41 to 51 to ox‐LDL.     

Structure of NADH peroxidase from Streptococcus faecalis 10C1 refined at 2.16 A resolution

The crystal structure of NADH peroxidase (EC 1.11.1.1) from Streptococcus faecalis 10C1 (Enterococcus faecalis) has been refined to a resolution of 2.16 A using the simulated annealing method. The final crystallographic R-factor is 17.7% for all data in the resolution range 7 to 2.16 A. The standard deviations are 0.015 A in bond lengths and 3.0 degrees in bond angles for the final model, which includes all 447 amino acid residues, one FAD and 369 water molecules. The enzyme is a symmetrical tetramer with point group D2; the symmetry is crystallographic. The redox center of the enzyme consists of FAD and a cysteine (Cys42), which forms a sulfenic acid (Cys-SOH) in its oxidized state. A histidine (His10) close to Cys42 is likely to act as an active-site base. In the analyzed crystal, the enzyme was in a non-native oxidation state with Cys42 oxidized to a sulfonic acid Cys-SO3H. The chain fold of NADH peroxidase is similar to those of disulfide oxidoreductases. A comparison with glutathione reductase, a representative of this enzyme family, is given.     

Characterization of the flavin association in hexose oxidase from Chondrus crispus

Hexose oxidase (EC 1.1.3.5) from Hansenula polymorpha was found to exhibit a dual covalent association of FAD with His79 via an 8 alpha-histidyl linkage as well as a covalent association between Cys138 and C-6 of the isoalloxazine moiety of FAD. Spectral properties of the wild-type enzyme exhibited maxima at 364 nm and 437 nm as well as a distinct shoulder at 445 nm. An H79K mutant enzyme exhibited only one maximum at 437 nm. The difference absorption spectrum between an oxidized and a substrate-reduced enzyme preparation showed maxima at 360 nm and 445 nm corresponding to an apparent novel type of association. Hexose oxidase showed a low, pH-independent fluorescence at 525 nm when excited at 450 nm. Flavin was released from the holoenzyme by treatment with trypsin. Sequencing of the flavopeptide revealed two peptides comprising positions 74-91 and 132-157 associated with FAD in equimolar amounts. A homology model of hexose oxidase was constructed using the crystal structure of glucooligosaccharide oxidase from Acremonium strictum as template. The model placed both of the sequences found above in the close vicinity of the FAD cofactor, and suggests covalent bonds between both His79 and Cys138 and FAD, in accordance with the chemical evidence. Based on the results, hexose oxidase is identified as incorporating FAD with a double covalent association with His79 and Cys138 in the holoenzyme. A reaction mechanism involving the concerted action of Tyr488 and Asp409 in hexose oxidase is suggested as the initiator of the proton abstraction from the substrate molecule in the active site.     

Three-dimensional structure of rat-liver acyl-CoA oxidase in complex with a fatty acid: insights into substrate-recognition and reactivity toward molecular oxygen

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.     

Insights into the reaction mechanism of glycosyl hydrolase family 49. Site-directed mutagenesis and substrate preference of isopullulanase.

Aspergillus niger isopullulanase (IPU) is the only pullulan-hydrolase in glycosyl hydrolase (GH) family 49 and does not hydrolyse dextran at all, while all other GH family 49 enzymes are dextran-hydrolysing enzymes. To investigate the common catalytic mechanism of GH family 49 enzymes, nine mutants were prepared to replace residues conserved among GH family 49 (four Trp, three Asp and two Glu). Homology modelling of IPU was also carried out based on the structure of Penicillium minioluteum dextranase, and the result showed that Asp353, Glu356, Asp372, Asp373 and Trp402, whose substitutions resulted in the reduction of activity for both pullulan and panose, were predicted to be located in the negatively numbered subsites. Three Asp-mutated enzymes, D353N, D372N and D373N, lost their activities, indicating that these residues are candidates for the catalytic residues of IPU. The W402F enzyme significantly reduced IPU activity, and the Km value was sixfold higher and the k0 value was 500-fold lower than those for the wild-type enzyme, suggesting that Trp402 is a residue participating in subsite -1. Trp31 and Glu273, whose substitutions caused a decrease in the activity for pullulan but not for panose, were predicted to be located in the interface between N-terminal and beta-helical domains. The substrate preference of the negatively numbered subsites of IPU resembles that of GH family 49 dextranases. These findings suggest that IPU and the GH family 49 dextranases have a similar catalytic mechanism in their negatively numbered subsites in spite of the difference of their substrate specificities.     

The structure of maize polyamine oxidase K300M mutant in complex with the natural substrates provides a snapshot of the catalytic mechanism of polyamine oxidation

Polyamine oxidases are FAD-dependent enzymes catalyzing the oxidation of polyamines at the secondary amino groups. Zea mays PAO (ZmPAO) oxidizes the carbon on the endo-side of the N5-nitrogen of spermidine (Spd) and spermine (Spm). The structure of ZmPAO revealed that the active site is formed by a catalytic tunnel in which the N5 atom of FAD lies in close proximity to the K300 side chain, the only active-site residue conserved in all PAOs. A water molecule, (HOH309), is hydrogen-bound to the amino group of K300 and mutation of this residue results in a 1400-fold decrease in the rate of flavin reduction. The structural studies on the catalytically impaired ZmPAO-K300M mutant described here show that substrates are bound in an 'out-of-register' mode and the HOH309 water molecule is absent in the enzyme-substrate complexes. Moreover, K300 mutation brings about a 60 mV decrease in the FAD redox potential and a 30-fold decrease in the FAD reoxidation rate, within a virtually unaltered geometry of the catalytic pocket. Taken together, these results indicate that the HOH309-K300 couple plays a major role in multiple steps of ZmPAO catalytic mechanism, such as correct substrate binding geometry as well as FAD reduction and reoxidation kinetics.     

A new genetic disorder in mitochondrial fatty acid beta-oxidation: ACAD9 deficiency

The acyl-CoA dehydrogenases are a family of multimeric flavoenzymes that catalyze the alpha,beta -dehydrogenation of acyl-CoA esters in fatty acid beta -oxidation and amino acid catabolism. Genetic defects have been identified in most of the acyl-CoA dehydrogenases in humans. Acyl-CoA dehydrogenase 9 (ACAD9) is a recently identified acyl-CoA dehydrogenase that demonstrates maximum activity with unsaturated long-chain acyl-CoAs. We now report three cases of ACAD9 deficiency. Patient 1 was a 14-year-old, previously healthy boy who died of a Reye-like episode and cerebellar stroke triggered by a mild viral illness and ingestion of aspirin. Patient 2 was a 10-year-old girl who first presented at age 4 mo with recurrent episodes of acute liver dysfunction and hypoglycemia, with otherwise minor illnesses. Patient 3 was a 4.5-year-old girl who died of cardiomyopathy and whose sibling also died of cardiomyopathy at age 21 mo. Mild chronic neurologic dysfunction was reported in all three patients. Defects in ACAD9 mRNA were identified in the first two patients, and all patients manifested marked defects in ACAD9 protein. Despite a significant overlap of substrate specificity, it appears that ACAD9 and very-long-chain acyl-CoA dehydrogenase are unable to compensate for each other in patients with either deficiency. Studies of the tissue distribution and gene regulation of ACAD9 and very-long-chain acyl-CoA dehydrogenase identify the presence of two independently regulated functional pathways for long-chain fat metabolism, indicating that these two enzymes are likely to be involved in different physiological functions.     

Role of the covalent flavin linkage in monomeric sarcosine oxidase

Monomeric sarcosine oxidase (MSOX) is a prototypical member of a recently recognized family of amine-oxidizing enzymes that all contain covalently bound flavin. Mutation of the covalent flavin attachment site in MSOX produces a catalytically inactive apoprotein (apoCys315Ala) that forms an unstable complex with FAD (K(d) = 100 muM), similar to that observed with wild-type apoMSOX where the complex is formed as an intermediate during covalent flavin attachment. In situ reconstitution of sarcosine oxidase activity is achieved by assaying apoCys315Ala in the presence of FAD or 8-nor-8-chloroFAD, an analogue with an approximately 55 mV higher reduction potential. After correction for an estimated 65% reconstitutable apoprotein, the specific activity of apoCys315Ala in the presence of excess FAD or 8-nor-8-chloroFAD is 14% or 80%, respectively, of that observed with wild-type MSOX. Unlike oxidized flavin, apoCys315Ala exhibits a high affinity for reduced flavin, as judged by results obtained with reduced 5-deazaFAD (5-deazaFADH(2)) where the estimated binding stoichiometry is unaffected by dialysis. The Cys315Ala.5-deazaFADH(2) complex is also air-stable but is readily oxidized by sarcosine imine, a reaction accompanied by release of weakly bound oxidized 5-deazaFAD. The dramatic difference in the binding affinity of apoCys315Ala for oxidized and reduced flavin indicates that the protein environment must induce a sizable increase in the reduction potential of noncovalently bound flavin (DeltaE(m) approximately 120 mV). The covalent flavin linkage prevents loss of weakly bound oxidized FAD and also modulates the flavin reduction potential in conjunction with the protein environment.     

Active site of Escherichia coli DNA photolyase: Asn378 is crucial both for stabilizing the neutral flavin radical cofactor and for DNA repair

Escherichia coli DNA photolyase repairs cyclobutane pyrimidine dimer (CPD) in UV-damaged DNA through a photoinduced electron transfer mechanism. The catalytic activity of the enzyme requires fully reduced FAD (FADH (-)). After purification in vitro, the cofactor FADH (-) in photolyase is oxidized into the neutral radical form FADH (*) under aerobic conditions and the enzyme loses its repair function. We have constructed a mutant photolyase in which asparagine 378 (N378) is replaced with serine (S). In comparison with wild-type photolyase, we found N378S mutant photolyase containing oxidized FAD (FAD ox) but not FADH (*) after routine purification procedures, but evidence shows that the mutant protein contains FADH (-) in vivo as the wild type. Although N378S mutant photolyase is photoreducable and capable of binding CPD in DNA, the activity assays indicate the mutant protein is catalytically inert. We conclude that the Asn378 residue of E. coli photolyase is crucial both for stabilizing the neutral flavin radical cofactor and for catalysis.     

The crystal structure of spermidine/spermine N1-acetyltransferase in complex with spermine provides insights into substrate binding and catalysis

The enzyme spermidine/spermine N (1)-acetyltransferase (SSAT) catalyzes the transfer of acetyl groups from acetylcoenzyme A to spermidine and spermine, as part of a polyamine degradation pathway. This work describes the crystal structure of SSAT in complex with coenzyme A, with and without bound spermine. The complex with spermine provides a direct view of substrate binding by an SSAT and demonstrates structural plasticity near the active site of the enzyme. Associated water molecules bridge several of the intermolecular contacts between spermine and the enzyme and form a "proton wire" between the side chain of Glu92 and the N1 amine of spermine. A single water molecule can also be seen forming hydrogen bonds with the side chains of Glu92, Asp93, and the N4 amine of spermine. Site-directed mutation of Glu92 to glutamine had a detrimental effect on both substrate binding and catalysis and shifted the optimal pH for enzyme activity further into alkaline solution conditions, while mutation of Asp93 to asparagine affected both substrate binding and catalysis without changing the pH dependence of the enzyme. Considered together, the structural and kinetic data suggest that Glu92 functions as a catalytic base to drive an otherwise unfavorable deprotonation step at physiological pH.     

Inactivation of two-electron reduced medium chain acyl-CoA dehydrogenase by 2-octynoyl-CoA

The acetylenic thioester, 2-octynoyl-CoA, inactivates medium chain acyl-CoA dehydrogenase from pig kidney by two distinct pathways depending on the redox state of the FAD prosthetic group. Inactivation of the oxidized dehydrogenase occurs with labeling of an active site glutamate residue and elimination of CoASH. Incubation of the reduced dehydrogenase with 2-octynoyl-CoA rapidly forms a kinetically stable dihydroflavin species which is resistant to reoxidation using trans-2-octenoyl-CoA, molecular oxygen, or electron transferring flavoprotein. The reduced enzyme derivative shows extensive bleaching at 446 nm with shoulders at 320 and 380 nm. Denaturation of the reduced derivative in 80% methanol yields a mixture of products which was characterized by HPLC, by uv/vis, and by radiolabeling experiments. Approximately 20% of the flavin is recovered as oxidized FAD, about 40% is retained covalently attached to the protein, and the remainder is distributed between several species eluting after FAD on reverse-phase HPLC. The spectrum of one of these species ressembles that of a N(5)-C(4a) dihydroflavin adduct. These data suggest that a primary reduced flavin species undergoes various rearrangements during release from the protein. The possibility that the inactive modified enzyme represents a covalent adduct between 2-octynoyl-CoA and reduced flavin is discussed. Analogous experiments using enzyme substituted with 1,5-dihydro-5-deaza-FAD show rapid and quantitative reoxidation of the flavin by 0.5 eq of 2-octynoyl-CoA.     

Crystal structures of Fms1 and its complex with spermine reveal substrate specificity

Fms1 is a rate-limiting enzyme for the biosynthesis of pantothenic acid in yeast. Fms1 has polyamine oxidase (PAO) activity, which converts spermine into spermidine and 3-aminopropanal. The 3-aminopropanal is further oxidized to produce beta-alanine, which is necessary for the biosynthesis of pantothenic acid. The crystal structures of Fms1 and its complex with the substrate spermine have been determined using the single-wavelength anomalous diffraction (SAD) phasing method. Fms1 consists of an FAD-binding domain, with Rossmann fold topology, and a substrate-binding domain. The active site is a tunnel located at the interface of the two domains. The substrate spermine binds to the active site mainly via hydrogen bonds and hydrophobic interactions. In the complex, C11 but not C9 of spermine is close enough to the catalytic site (N5 of FAD) to be oxidized. Therefore, the products are spermidine and 3-aminopropanal, rather than 3-(aminopropyl) 4-aminobutyraldehyde and 1,3-diaminoprone.     

Substrate binding to histone deacetylases as shown by the crystal structure of the HDAC8-substrate complex

Histone deacetylases (HDACs)-an enzyme family that deacetylates histones and non-histone proteins-are implicated in human diseases such as cancer, and the first-generation of HDAC inhibitors are now in clinical trials. Here, we report the 2.0 A resolution crystal structure of a catalytically inactive HDAC8 active-site mutant, Tyr306Phe, bound to an acetylated peptidic substrate. The structure clarifies the role of active-site residues in the deacetylation reaction and substrate recognition. Notably, the structure shows the unexpected role of a conserved residue at the active-site rim, Asp 101, in positioning the substrate by directly interacting with the peptidic backbone and imposing a constrained cis-conformation. A similar interaction is observed in a new hydroxamate inhibitor-HDAC8 structure that we also solved. The crucial role of Asp 101 in substrate and inhibitor recognition was confirmed by activity and binding assays of wild-type HDAC8 and Asp101Ala, Tyr306Phe and Asp101Ala/Tyr306Phe mutants.     

Fourier-transform infrared study of the photoactivation process of Xenopus (6-4) photolyase

Photolyases (PHRs) are blue light-activated DNA repair enzymes that maintain genetic integrity by reverting UV-induced photoproducts into normal bases. The flavin adenine dinucleotide (FAD) chromophore of PHRs has four different redox states: oxidized (FAD(ox)), anion radical (FAD(?-)), neutral radical (FADH(?)), and fully reduced (FADH(-)). We combined difference Fourier-transform infrared (FTIR) spectroscopy with UV-visible spectroscopy to study the detailed photoactivation process of Xenopus (6-4) PHR. Two photons produce the enzymatically active, fully reduced PHR from oxidized FAD: FAD(ox) is converted to semiquinone via light-induced one-electron and one-proton transfers and then to FADH(-) by light-induced one-electron transfer. We successfully trapped FAD(?-) at 200 K, where electron transfer occurs but proton transfer does not. UV-visible spectroscopy following 450 nm illumination of FAD(ox) at 277 K defined the FADH(?)/FADH(-) mixture and allowed calculation of difference FTIR spectra among the four redox states. The absence of a characteristic C=O stretching vibration indicated that the proton donor is not a protonated carboxylic acid. Structural changes in Trp and Tyr are suggested by UV-visible and FTIR analysis of FAD(?-) at 200 K. Spectral analysis of amide I vibrations revealed structural perturbation of the protein's β-sheet during initial electron transfer (FAD(?-) formation), a transient increase in α-helicity during proton transfer (FADH(?) formation), and reversion to the initial amide I signal following subsequent electron transfer (FADH(-) formation). Consequently, in (6-4) PHR, unlike cryptochrome-DASH, formation of enzymatically active FADH(-) did not perturb α-helicity. Protein structural changes in the photoactivation of (6-4) PHR are discussed on the basis of these FTIR observations.     

Acyl-CoA Dehydrogenases: Dynamic History of Protein Family Evolution

The acyl-CoA dehydrogenases (ACADs) are enzymes that catalyze the α,β-dehydrogenation of acyl-CoA esters in fatty acid and amino acid catabolism. Eleven ACADs are now recognized in the sequenced human genome, and several homologs have been reported from bacteria, fungi, plants, and nematodes. We performed a systematic comparative genomic study, integrating homology searches with methods of phylogenetic reconstruction, to investigate the evolutionary history of this family. Sequence analyses indicate origin of the family in the common ancestor of Archaea, Bacteria, and Eukaryota, illustrating its essential role in the metabolism of early life. At least three ACADs were already present at that time: ancestral glutaryl-CoA dehydrogenase (GCD), isovaleryl-CoA dehydrogenase (IVD), and ACAD10/11. Two gene duplications were unique to the eukaryotic domain: one resulted in the VLCAD and ACAD9 paralogs and another in the ACAD10 and ACAD11 paralogs. The overall patchy distribution of specific ACADs across the tree of life is the result of dynamic evolution that includes numerous rounds of gene duplication and secondary losses, interdomain lateral gene transfer events, alteration of cellular localization, and evolution of novel proteins by domain acquisition. Our finding that eukaryotic ACAD species are more closely related to bacterial ACADs is consistent with endosymbiotic origin of ACADs in eukaryotes and further supported by the localization of all nine previously studied ACADs in mitochondria. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.