The flavoprotein WrbA from Escherichia coli is considered to be the prototype of a new family of multimeric flavodoxin-like proteins that are implicated in cell protection against oxidative stress. The present study is aimed at structural characterization of the E. coli protein with respect to its recently revealed oxidoreductase activity. Crystals of WrbA holoprotein in complex with the oxidized flavin cofactor (FMN) were obtained using standard vapour-diffusion techniques. Deep yellow tetragonal crystals obtained from differing crystallization conditions display different space groups and unit-cell parameters. X-ray crystal structures of the WrbA holoprotein have been determined to resolutions of 2.0 and 2.6 A.
- MeSH
- DNA-Binding Proteins chemistry metabolism MeSH
- Escherichia coli metabolism MeSH
- Financing, Organized MeSH
- Flavin Mononucleotide chemistry metabolism MeSH
- Crystallization MeSH
- Crystallography, X-Ray MeSH
- Escherichia coli Proteins chemistry metabolism MeSH
- Repressor Proteins chemistry metabolism MeSH
The activity of the light-oxygen-voltage/helix-turn-helix (LOV-HTH) photoreceptor EL222 is regulated through protein-protein and protein-DNA interactions, both triggered by photo-excitation of its flavin mononucleotide (FMN) cofactor. To gain molecular-level insight into the photocycle of EL222, we applied complementary methods: macromolecular X-ray crystallography (MX), nuclear magnetic resonance (NMR) spectroscopy, optical spectroscopies (infrared and UV-visible), molecular dynamics/metadynamics (MD/metaD) simulations, and protein engineering using noncanonical amino acids. Kinetic experiments provided evidence for two distinct EL222 conformations (lit1 and lit2) that become sequentially populated under illumination. These two lit states were assigned to covalently bound N5 protonated, and noncovalently bound hydroquinone forms of FMN, respectively. Only subtle structural differences were observed between the monomeric forms of all three EL222 species (dark, lit1, and lit2). While the dark state is largely monomeric, both lit states undergo monomer-dimer exchange. Furthermore, molecular modeling revealed differential dynamics and interdomain separation times arising from the three FMN states (oxidized, adduct, and reduced). Unexpectedly, all three EL222 species can associate with DNA, but only upon blue-light irradiation, a high population of stable complexes is obtained. Overall, we propose a model of EL222 activation where photoinduced changes in the FMN moiety shift the population equilibrium toward an open conformation that favors self-association and DNA-binding.
- MeSH
- Bacterial Proteins chemistry metabolism MeSH
- DNA-Binding Proteins chemistry metabolism MeSH
- DNA * chemistry metabolism MeSH
- Flavin Mononucleotide * chemistry metabolism MeSH
- Flavins chemistry metabolism MeSH
- Kinetics MeSH
- Protein Conformation MeSH
- Crystallography, X-Ray MeSH
- Oxidation-Reduction * MeSH
- Molecular Dynamics Simulation MeSH
- Light * MeSH
- Thermosynechococcus metabolism MeSH
- Transcription Factors metabolism chemistry MeSH
- Protein Binding MeSH
- Publication type
- Journal Article MeSH
Ferric reductase B (FerB) is a flavin mononucleotide (FMN)-containing NAD(P)H:acceptor oxidoreductase structurally close to the Gluconacetobacter hansenii chromate reductase (ChrR). The crystal structure of ChrR was previously determined with a chloride bound proximal to FMN in the vicinity of Arg101, and the authors suggested that the anionic electron acceptors, chromate and uranyl tricarbonate, bind similarly. Here, we identify the corresponding arginine residue in FerB (Arg95) as being important for the reaction of FerB with superoxide. Four mutants at position 95 were prepared and found kinetically to have impaired capacity for superoxide binding. Stopped-flow data for the flavin cofactor showed that the oxidative step is rate limiting for catalytic turnover. The findings are consistent with a role for FerB as a superoxide scavenging contributor.
- MeSH
- Arginine genetics MeSH
- Flavin Mononucleotide chemistry genetics MeSH
- Flavins genetics metabolism MeSH
- FMN Reductase chemistry genetics MeSH
- Catalytic Domain genetics MeSH
- Kinetics MeSH
- Protein Conformation * MeSH
- Crystallography, X-Ray MeSH
- Oxidation-Reduction MeSH
- Oxidoreductases chemistry genetics MeSH
- Paracoccus denitrificans chemistry enzymology MeSH
- Amino Acid Sequence genetics MeSH
- Superoxides metabolism MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
FerB from Paracoccus denitrificans is a soluble cytoplasmic flavoprotein that accepts redox equivalents from NADH or NADPH and transfers them to various acceptors such as quinones, ferric complexes and chromate. The crystal structure and small-angle X-ray scattering measurements in solution reported here reveal a head-to-tail dimer with two flavin mononucleotide groups bound at the opposite sides of the subunit interface. The dimers tend to self-associate to a tetrameric form at higher protein concentrations. Amino acid residues important for the binding of FMN and NADH and for the catalytic activity are identified and verified by site-directed mutagenesis. In particular, we show that Glu77 anchors a conserved water molecule in close proximity to the O2 of FMN, with the probable role of facilitating flavin reduction. Hydride transfer is shown to occur from the 4-pro-S position of NADH to the solvent-accessible si side of the flavin ring. When using deuterated NADH, this process exhibits a kinetic isotope effect of about 6 just as does the NADH-dependent quinone reductase activity of FerB; the first, reductive half-reaction of flavin cofactor is thus rate-limiting. Replacing the bulky Arg95 in the vicinity of the active site with alanine substantially enhances the activity towards external flavins that obeys the standard bi-bi ping-pong reaction mechanism. The new evidence for a cryptic flavin reductase activity of FerB justifies the previous inclusion of this enzyme in the protein family of NADPH-dependent FMN reductases.
- MeSH
- Amino Acids chemistry genetics metabolism MeSH
- Bacterial Proteins chemistry genetics metabolism MeSH
- Biocatalysis MeSH
- X-Ray Diffraction MeSH
- Flavin Mononucleotide chemistry metabolism MeSH
- Flavins chemistry metabolism MeSH
- Flavoproteins chemistry genetics metabolism MeSH
- Catalytic Domain genetics MeSH
- Kinetics MeSH
- Crystallography, X-Ray MeSH
- Scattering, Small Angle MeSH
- Models, Molecular MeSH
- Molecular Sequence Data MeSH
- Protein Multimerization MeSH
- Mutagenesis, Site-Directed MeSH
- NADH, NADPH Oxidoreductases chemistry classification metabolism MeSH
- NADP chemistry metabolism MeSH
- Oxidation-Reduction MeSH
- Paracoccus denitrificans enzymology genetics MeSH
- Amino Acid Sequence MeSH
- Sequence Homology, Amino Acid MeSH
- Protein Structure, Tertiary * MeSH
- Protein Binding MeSH
- Binding Sites genetics MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
The Escherichia coli protein WrbA, an FMN-dependent NAD(P)H:quinone oxidoreductase, was crystallized under new conditions in the presence of FAD or the native cofactor FMN. Slow-growing deep yellow crystals formed with FAD display the tetragonal bipyramidal shape typical for WrbA and diffract to 1.2 Å resolution, the highest yet reported. Faster-growing deep yellow crystals formed with FMN display an atypical shape, but diffract to only ∼1.6 Å resolution and are not analysed further here. The 1.2 Å resolution structure detailed here revealed only FMN in the active site and no electron density that can accommodate the missing parts of FAD. The very high resolution supports the modelling of the FMN isoalloxazine with a small but distinct propeller twist, apparently the first experimental observation of this predicted conformation, which appears to be enforced by the protein through a network of hydrogen bonds. Comparison of the electron density of the twisted isoalloxazine ring with the results of QM/MM simulations is compatible with the oxidized redox state. The very high resolution also supports the unique refinement of Met10 as the sulfoxide, confirmed by mass spectrometry. Bond lengths, intramolecular distances, and the pattern of hydrogen-bond donors and acceptors suggest the cofactor may interact with Met10. Slow incorporation of FMN, which is present as a trace contaminant in stocks of FAD, into growing crystals may be responsible for the near-atomic resolution, but a direct effect of the conformation of FMN and/or Met10 sulfoxide cannot be ruled out.
- MeSH
- X-Ray Diffraction MeSH
- Flavin-Adenine Dinucleotide chemistry metabolism MeSH
- Flavin Mononucleotide chemistry metabolism MeSH
- Crystallization MeSH
- Crystallography, X-Ray MeSH
- NAD(P)H Dehydrogenase (Quinone) chemistry metabolism MeSH
- Oxidation-Reduction MeSH
- Escherichia coli Proteins chemistry metabolism MeSH
- Repressor Proteins chemistry metabolism MeSH
- Protein Binding MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
- Research Support, U.S. Gov't, Non-P.H.S. MeSH
Old Yellow Enzymes (OYEs) are NAD(P)H dehydrogenases of not fully resolved physiological roles that are widespread among bacteria, plants, and fungi and have a great potential for biotechnological applications. We determined the apo form crystal structure of a member of the OYE class, glycerol trinitrate reductase XdpB, from Agrobacterium bohemicum R89-1 at 2.1 Å resolution. In agreement with the structures of the related bacterial OYEs, the structure revealed the TIM barrel fold with an N-terminal β-hairpin lid, but surprisingly, the structure did not contain its cofactor FMN. Its putative binding site was occupied by a pentapeptide TTSDN from the C-terminus of a symmetry related molecule. Biochemical experiments confirmed a specific concentration-dependent oligomerization and a low FMN content. The blocking of the FMN binding site can exist in vivo and regulates enzyme activity. Our bioinformatic analysis indicated that a similar self-inhibition could be expected in more OYEs which we designated as subgroup OYE C1. This subgroup is widespread among G-bacteria and can be recognized by the conserved sequence GxxDYP in proximity of the C termini. In proteobacteria, the C1 subgroup OYEs are typically coded in one operon with short-chain dehydrogenase. This operon is controlled by the tetR-like transcriptional regulator. OYEs coded in these operons are unlikely to be involved in the oxidative stress response as the other known members of the OYE family because no upregulation of XdpB was observed after exposing A. bohemicum R89-1 to oxidative stress.
- MeSH
- Agrobacterium enzymology genetics MeSH
- Genes, Bacterial MeSH
- Bacterial Proteins chemistry genetics metabolism MeSH
- Flavin Mononucleotide metabolism MeSH
- Catalytic Domain MeSH
- Kinetics MeSH
- Crystallography, X-Ray MeSH
- Protein Structure, Quaternary MeSH
- Models, Molecular MeSH
- NADPH Dehydrogenase chemistry genetics metabolism MeSH
- Operon MeSH
- Oxidative Stress MeSH
- Oxidoreductases chemistry genetics metabolism MeSH
- Computational Biology MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
Glucose oxidase (GOX) is a homodimeric glycoprotein with tightly bound one molecule of FAD cofactor per monomer of the protein. GOX has numerous applications, but the preparation of biotechnologically interesting GOX sensors requires a removal of the native FAD cofactor. This process often leads to unwanted irreversible deflavination and, as a consequence, to the low enzyme recovery. Molecular mechanisms of reversible reflavination are poorly understood; our current knowledge is based only on empiric rules, which is clearly insufficient for further development. To develop conceptual understanding of flavin-binding competent states, we studied the effect of deflavination protocols on conformational properties of GOX. After deflavination, the apoform assembles into soluble oligomers with nearly native-like holoform secondary structure but largely destabilized tertiary structure presumambly due to the packing density defects around the vacant flavin binding site. The reflavination is cooperative but not fully efficient; after the binding the flavin cofactor, the protein directly disassembles into native homodimers while the fraction of oligomers remains irreversibly inactivated. Importantly, the effect of Hofmeister salts on the conformational properties of GOX and reflavination efficiency indicates that the native-like residual tertiary structure in the molten-globule states favorably supports the reflavination and minimizes the inactivated oligomers. We interpret our results by combining the ligand-induced changes in quaternary structure with salt-sensitive, non-equilibrated conformational selection model. In summary, our work provides the very first steps toward molecular understanding the complexity of the GOX reflavination mechanism.
- MeSH
- Aspergillus niger enzymology MeSH
- Biocatalysis MeSH
- Circular Dichroism MeSH
- Calorimetry, Differential Scanning MeSH
- Flavin-Adenine Dinucleotide chemistry metabolism MeSH
- Glucose Oxidase chemistry metabolism MeSH
- Protein Multimerization MeSH
- Protein Isoforms chemistry metabolism MeSH
- Protein Structure, Secondary MeSH
- Spectrophotometry, Ultraviolet MeSH
- Protein Stability MeSH
- Temperature MeSH
- Protein Structure, Tertiary MeSH
- Publication type
- Journal Article MeSH
The flavoenzyme cytokinin dehydrogenase (CKX) catalyzes an irreversible deactivation of plant hormones cytokinins through oxidative cleavage of the cytokinin side chain to yield adenine or adenosine and an aldehyde. In the catalytic cycle of CKX, the cytokinin-reduced flavin cofactor is reoxidized by a suitable electron acceptor. We have recently demonstrated that the oxidation products of natural hydroxamic acid 2,4-dihydroxy-7-methoxy-1,4-benzoxazin-3-one (DIMBOA) function as effective electron acceptors of apoplastic CKX from maize. The stable oxidation product of DIMBOA reacting with peroxidase or laccase was identified as 4-nitrosoresorcinol 1-monomethyl ether (coniferron), which, however, is only a weak electron acceptor of CKX. Further analyses suggested formation of transient free radicals that were estimated to reoxidize the cytokinin-reduced flavin cofactor of CKX with the rates comparable to those of flavin reduction.
5,10-Methylenetetrahydrofolate reductase (MTHFR) deficiency is the most common inherited disorder of folate metabolism and causes severe hyperhomocysteinaemia. To better understand the relationship between mutation and function, we performed molecular genetic analysis of 76 MTHFR deficient patients, followed by extensive enzymatic characterization of fibroblasts from 72 of these. A deleterious mutation was detected on each of the 152 patient alleles, with one allele harboring two mutations. Sixty five different mutations (42 novel) were detected, including a common splicing mutation (c.1542G>A) found in 21 alleles. Using an enzyme assay in the physiological direction, we found residual activity (1.7%-42% of control) in 42 cell lines, of which 28 showed reduced affinity for nicotinamide adenine dinucleotide phosphate (NADPH), one reduced affinity for methylenetetrahydrofolate, five flavin adenine dinucleotide-responsiveness, and 24 abnormal kinetics of S-adenosylmethionine inhibition. Missense mutations causing virtually absent activity were found exclusively in the N-terminal catalytic domain, whereas missense mutations in the C-terminal regulatory domain caused decreased NADPH binding and disturbed inhibition by S-adenosylmethionine. Characterization of patients in this way provides a basis for improved diagnosis using expanded enzymatic criteria, increases understanding of the molecular basis of MTHFR dysfunction, and points to the possible role of cofactor or substrate in the treatment of patients with specific mutations.
- MeSH
- Enzyme Activation MeSH
- Alleles MeSH
- Alternative Splicing MeSH
- Exons MeSH
- Fibroblasts metabolism MeSH
- Genetic Association Studies * MeSH
- Homocystinuria diagnosis genetics metabolism MeSH
- Introns MeSH
- Polymorphism, Single Nucleotide MeSH
- Kinetics MeSH
- Humans MeSH
- Methylenetetrahydrofolate Reductase (NADPH2) deficiency genetics metabolism MeSH
- Mutation MeSH
- Psychotic Disorders diagnosis genetics metabolism MeSH
- Protein Stability MeSH
- Muscle Spasticity diagnosis genetics metabolism MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
Berberine bridge enzyme-like (BBE-like) proteins form a multigene family (pfam 08031), which is present in plants, fungi and bacteria. They adopt the vanillyl alcohol-oxidase fold and predominantly show bi-covalent tethering of the FAD cofactor to a cysteine and histidine residue, respectively. The Arabidopsis thaliana genome was recently shown to contain genes coding for 28 BBE-like proteins, while featuring four distinct active site compositions. We determined the structure of a member of the AtBBE-like protein family (termed AtBBE-like 28), which has an active site composition that has not been structurally and biochemically characterized thus far. The most salient and distinguishing features of the active site found in AtBBE-like 28 are a mono-covalent linkage of a histidine to the 8α-position of the flavin-isoalloxazine ring and the lack of a second covalent linkage to the 6-position, owing to the replacement of a cysteine with a histidine. In addition, the structure reveals the interaction of a glutamic acid (Glu426) with an aspartic acid (Asp369) at the active site, which appear to share a proton. This arrangement leads to the delocalization of a negative charge at the active site that may be exploited for catalysis. The structure also indicates a shift of the position of the isoalloxazine ring in comparison to other members of the BBE-like family. The dioxygen surrogate chloride was found near the C(4a) position of the isoalloxazine ring in the oxygen pocket, pointing to a rapid reoxidation of reduced enzyme by dioxygen. A T-DNA insertional mutant line for AtBBE-like 28 results in a phenotype, that is characterized by reduced biomass and lower salt stress tolerance. Multiple sequence analysis showed that the active site composition found in AtBBE-like 28 is only present in the Brassicaceae, suggesting that it plays a specific role in the metabolism of this plant family.
- MeSH
- Arabidopsis enzymology genetics MeSH
- Species Specificity MeSH
- Catalytic Domain MeSH
- Aspartic Acid chemistry genetics MeSH
- Glutamic Acid chemistry genetics MeSH
- Mutagenesis MeSH
- Oxidoreductases, N-Demethylating chemistry genetics MeSH
- Arabidopsis Proteins chemistry genetics MeSH
- Protein Structure, Secondary MeSH
- Salt Tolerance physiology MeSH
- Publication type
- Journal Article MeSH
