ABSTRACT
[NiFe]-hydrogenase from Desulfovibrio vulgaris Miyazaki F is an O2-sensitive enzyme that is inactivated in the presence of O2 but the oxidized enzyme can recover its catalytic activity by reacting with H2 under anaerobic conditions. Here, we report the first neutron structure of [NiFe]-hydrogenase in its oxidized state, determined at a resolution of 2.20 Å. This resolution allowed us to reinvestigate the structure of the oxidized active site and to observe the positions of protons in several short hydrogen bonds. X-ray anomalous scattering data revealed that a part of the Ni ion is dissociated from the active site Ni-Fe complex and forms a new square-planar Ni complex, accompanied by rearrangement of the coordinated thiolate ligands. One of the thiolate Sγ atoms is oxidized to a sulfenate anion but remains attached to the Ni ion, which was evaluated by quantum chemical calculations. These results suggest that the square-planar complex can be generated by the attack of reactive oxygen species derived from O2, as distinct from one-electron oxidation leading to a conventional oxidized form of the Ni-Fe complex. Another major finding of this neutron structure analysis is that the Cys17S thiolate Sγ atom coordinating to the proximal Fe-S cluster forms an unusual hydrogen bond with the main-chain amide N atom of Gly19S with a distance of 3.25 Å, where the amide proton appears to be delocalized between the donor and acceptor atoms. This observation provides insight into the contribution of the coordinated thiolate ligands to the redox reaction of the Fe-S cluster.
ABSTRACT
Protein crystals are generally fragile and sensitive to subtle changes such as pH, ionic strength, and/or temperature in their crystallization mother liquor. Here, using T4 phage lysozyme as a model protein, the three-dimensional rigidification of protein crystals was conducted by introducing disulfide cross-links between neighboring molecules in the crystal. The effect of cross-linking on the stability of the crystals was evaluated by microscopic observation and X-ray diffraction. When soaking the obtained cross-linked crystals into a precipitant-free solution, the crystals held their shape without dissolution and diffracted to approximately 1.1 Å resolution, comparable to that of the non-cross-linked crystals. Such cross-linked crystals maintained their diffraction even when immersed in other solutions with pH values from 4 to 10, indicating that the disulfide cross-linking made the packing contacts enforced and resulted in some mechanical strength in response to changes in the preservation conditions. Furthermore, the cross-linked crystals gained stability to permit soaking into solutions containing high concentrations of organic solvents. The results suggest the possibility of obtaining protein crystals for effective drug screening by introducing appropriate cross-linked disulfide bonds.
ABSTRACT
SignificanceDNA damage causes loss of or alterations in genetic information, resulting in cell death or mutations. Ionizing radiations produce local, multiple DNA damage sites called clustered DNA damage. In this study, a complete protocol was established to analyze the damage complexity of clustered DNA damage, wherein damage-containing genomic DNA fragments were selectively concentrated via pulldown, and clustered DNA damage was visualized by atomic force microscopy. It was found that X-rays and Fe ion beams caused clustered DNA damage. Fe ion beams also produced clustered DNA damage with high complexity. Fe ion beam-induced complex DNA double-strand breaks (DSBs) containing one or more base lesion(s) near the DSB end were refractory to repair, implying their lethal effects.
Subject(s)
DNA Damage , Radiation, Ionizing , DNA/genetics , DNA/radiation effects , DNA Breaks, Double-Stranded , DNA Repair , Microscopy, Atomic ForceABSTRACT
Vibrio vulnificus is a Gram-negative pathogenic bacterium that causes serious infections in humans and requires iron for growth. A clinical isolate, V. vulnificus M2799, secretes a catecholate siderophore, vulnibactin, that captures ferric ions from the environment. In the ferric-utilization system in V. vulnificus M2799, an isochorismate synthase (ICS) and an outer membrane receptor, VuuA, are required under low-iron conditions, but alternative proteins FatB and VuuB can function as a periplasmic-binding protein and a ferric-chelate reductase, respectively. The vulnibactin-export system is assembled from TolCV1 and several RND proteins, including VV1_1681. In heme acquisition, HupA and HvtA serve as specific outer membrane receptors and HupB is a sole periplasmic-binding protein, unlike FatB in the ferric-vulnibactin utilization system. We propose that ferric-siderophore periplasmic-binding proteins and ferric-chelate reductases are potential targets for drug discovery in infectious diseases.
Subject(s)
Iron/metabolism , Vibrio vulnificus/metabolism , Animals , Aquatic Organisms , Ions , Periplasmic Binding Proteins/metabolism , Vibrio vulnificus/geneticsABSTRACT
A membrane-bound hydrogenase from Desulfovibrio vulgaris Miyazaki F is a metalloenzyme that contains a binuclear Ni-Fe complex in its active site and mainly catalyzes the oxidation of molecular hydrogen to generate a proton gradient in the bacterium. The active-site Ni-Fe complex of the aerobically purified enzyme shows its inactive oxidized form, which can be reactivated through reduction by hydrogen. Here, in order to understand how the oxidized form is reactivated by hydrogen and further to directly evaluate the bridging of a hydride ligand in the reduced form of the Ni-Fe complex, a neutron structure determination was undertaken on single crystals grown in a hydrogen atmosphere. Cryogenic crystallography is being introduced into the neutron diffraction research field as it enables the trapping of short-lived intermediates and the collection of diffraction data to higher resolution. To optimize the cooling of large crystals under anaerobic conditions, the effects on crystal quality were evaluated by X-rays using two typical methods, the use of a cold nitrogen-gas stream and plunge-cooling into liquid nitrogen, and the former was found to be more effective in cooling the crystals uniformly than the latter. Neutron diffraction data for the reactivated enzyme were collected at the Japan Photon Accelerator Research Complex under cryogenic conditions, where the crystal diffracted to a resolution of 2.0â Å. A neutron diffraction experiment on the reduced form was carried out at Oak Ridge National Laboratory under cryogenic conditions and showed diffraction peaks to a resolution of 2.4â Å.
Subject(s)
Crystallography/methods , Hydrogenase/chemistry , Neutron Diffraction/methods , Desulfovibrio vulgaris/enzymology , Freezing , Models, MolecularABSTRACT
Cytochrome c oxidase (CcO) reduces O2 to water, coupled with a proton-pumping process. The structure of the O2-reduction site of CcO contains two reducing equivalents, Fe a32+ and CuB1+, and suggests that a peroxide-bound state (Fe a33+-O--O--CuB2+) rather than an O2-bound state (Fe a32+-O2) is the initial catalytic intermediate. Unexpectedly, however, resonance Raman spectroscopy results have shown that the initial intermediate is Fe a32+-O2, whereas Fe a33+-O--O--CuB2+ is undetectable. Based on X-ray structures of static noncatalytic CcO forms and mutation analyses for bovine CcO, a proton-pumping mechanism has been proposed. It involves a proton-conducting pathway (the H-pathway) comprising a tandem hydrogen-bond network and a water channel located between the N- and P-side surfaces. However, a system for unidirectional proton-transport has not been experimentally identified. Here, an essentially identical X-ray structure for the two catalytic intermediates (P and F) of bovine CcO was determined at 1.8 Å resolution. A 1.70 Å Fe-O distance of the ferryl center could best be described as Fe a34+ = O2-, not as Fe a34+-OH- The distance suggests an â¼800-cm-1 Raman stretching band. We found an interstitial water molecule that could trigger a rapid proton-coupled electron transfer from tyrosine-OH to the slowly forming Fe a33+-O--O--CuB2+ state, preventing its detection, consistent with the unexpected Raman results. The H-pathway structures of both intermediates indicated that during proton-pumping from the hydrogen-bond network to the P-side, a transmembrane helix closes the water channel connecting the N-side with the hydrogen-bond network, facilitating unidirectional proton-pumping during the P-to-F transition.
Subject(s)
Electron Transport Complex IV/metabolism , Oxygen/metabolism , Animals , Catalytic Domain , Cattle , Crystallography, X-Ray , Electron Transport Complex IV/chemistry , Models, Molecular , Oxidation-Reduction , Protein Conformation , Protein Subunits/chemistry , Protein Subunits/metabolism , ProtonsABSTRACT
Hydrogenase is a key enzyme for a coming hydrogen energy society, because it has strong catalytic activities on both uptake and production of dihydrogen. We, however, have to overcome the sensitivity against O2 of the enzyme, because hydrogenase is, generally, easily inactivated in the presence of O2. In this study, we have revisited the crystal structures of [NiFe]hydrogenase from sulfate-reducing bacterium in the several oxidized and reduced conditions. Our results revealed that the Ni-Fe active site of the enzyme exposed into O2 showed two forms, Form-1 and Form-2. The Ni-Fe active site in Form-1 showed the typical Ni-B (inactive ready) structure, whereas those in Form-2 lost Ni with no relation to an exposure time to O2, and two cysteinyl sulfur ligands made a disulfide bond. On the other hand, the formation of sulfenylation of the cysteinyl ligand to Ni, which is often observed in the oxidized form, did not correlate with the Ni-elimination, but with exposure time to O2.
Subject(s)
Hydrogenase/chemistry , Nickel/chemistry , Oxygen/chemistry , Catalytic Domain , Crystallography, X-Ray , Cysteine/chemistry , Ligands , Molecular Structure , Oxidation-ReductionABSTRACT
T4 phage lysozyme is an inverting glycoside hydrolase that degrades the murein of bacterial cell walls by cleaving the ß-1,4-glycosidic bond. The substitution of the catalytic Thr26 residue to a histidine converts the wild type from an inverting to a retaining enzyme, which implies that the original general acid Glu11 can also act as an acid/base catalyst in the hydrolysis. Here, we have determined the neutron structure of the perdeuterated T26H mutant to clarify the protonation states of Glu11 and the substituted His26, which are key in the retaining reaction. The 2.09-Å resolution structure shows that the imidazole group of His26 is in its singly protonated form in the active site, suggesting that the deprotonated NÉ2 atom of His26 can attack the anomeric carbon of bound substrate as a nucleophile. The carboxyl group of Glu11 is partially protonated and interacts with the unusual neutral state of the guanidine moiety of Arg145, as well as two heavy water molecules. Considering that one of the water-binding sites has the potential to be occupied by a hydronium ion, the bulk solvent could be the source for the protonation of Glu11. The respective protonation states of Glu11 and His26 are consistent with the bond lengths determined by an unrestrained refinement of the high-resolution X-ray structure of T26H at 1.04-Å resolution. The detail structural information, including the coordinates of the deuterium atoms in the active site, provides insight into the distinctively different catalytic activities of the mutant and wild type enzymes.
Subject(s)
Bacteriophage T4/enzymology , Muramidase/metabolism , Muramidase/ultrastructure , Bacteriophage T4/genetics , Binding Sites/genetics , Crystallography, X-Ray , Hydrolysis , Models, Molecular , Muramidase/chemistry , Muramidase/genetics , Mutation/genetics , Neutrons , Protein ConformationABSTRACT
UDP-glucose: anthocyanidin 3-O-glucosyltransferase (UGT78K6) from Clitoria ternatea catalyzes the transfer of glucose from UDP-glucose to anthocyanidins such as delphinidin. After the acylation of the 3-O-glucosyl residue, the 3'- and 5'-hydroxyl groups of the product are further glucosylated by a glucosyltransferase in the biosynthesis of ternatins, which are anthocyanin pigments. To understand the acceptor-recognition scheme of UGT78K6, the crystal structure of UGT78K6 and its complex forms with anthocyanidin delphinidin and petunidin, and flavonol kaempferol were determined to resolutions of 1.85 Å, 2.55 Å, 2.70 Å, and 1.75 Å, respectively. The enzyme recognition of unstable anthocyanidin aglycones was initially observed in this structural determination. The anthocyanidin- and flavonol-acceptor binding details are almost identical in each complex structure, although the glucosylation activities against each acceptor were significantly different. The 3-hydroxyl groups of the acceptor substrates were located at hydrogen-bonding distances to the Nε2 atom of the His17 catalytic residue, supporting a role for glucosyl transfer to the 3-hydroxyl groups of anthocyanidins and flavonols. However, the molecular orientations of these three acceptors are different from those of the known flavonoid glycosyltransferases, VvGT1 and UGT78G1. The acceptor substrates in UGT78K6 are reversely bound to its binding site by a 180° rotation about the O1-O3 axis of the flavonoid backbones observed in VvGT1 and UGT78G1; consequently, the 5- and 7-hydroxyl groups are protected from glucosylation. These substrate recognition schemes are useful to understand the unique reaction mechanism of UGT78K6 for the ternatin biosynthesis, and suggest the potential for controlled synthesis of natural pigments.
Subject(s)
Anthocyanins/chemistry , Clitoria/enzymology , Glucosyltransferases/chemistry , Plant Proteins/chemistry , Uridine Diphosphate Glucose/chemistry , Anthocyanins/metabolism , Binding Sites , Clitoria/chemistry , Glucosyltransferases/metabolism , Models, Molecular , Plant Proteins/metabolism , Substrate Specificity , Uridine Diphosphate Glucose/metabolismABSTRACT
Proteasome formation does not occur due to spontaneous self-organization but results from a highly ordered process assisted by several assembly chaperones. The assembly of the proteasome ATPase subunits is assisted by four client-specific chaperones, of which three have been structurally resolved. Here, we provide the structural basis for the working mechanisms of the last, hereto structurally uncharacterized assembly chaperone, Nas2. We revealed that Nas2 binds to the Rpt5 subunit in a bivalent mode: the N-terminal helical domain of Nas2 masks the Rpt1-interacting surface of Rpt5, whereas its C-terminal PDZ domain caps the C-terminal proteasome-activating motif. Thus, Nas2 operates as a proteasome activation blocker, offering a checkpoint during the formation of the 19S ATPase prior to its docking onto the proteolytic 20S core particle.
Subject(s)
Adenosine Triphosphatases/chemistry , Adenosine Triphosphatases/metabolism , Proteasome Endopeptidase Complex/metabolism , Saccharomyces cerevisiae Proteins/chemistry , Saccharomyces cerevisiae Proteins/metabolism , Adenosine Triphosphatases/genetics , Crystallography, X-Ray , Models, Molecular , Molecular Chaperones/chemistry , Molecular Chaperones/metabolism , Mutation , PDZ Domains , Protein Structure, Tertiary , Saccharomyces cerevisiae Proteins/genetics , Surface Plasmon ResonanceABSTRACT
Flowers of the butterfly pea (Clitoria ternatea) accumulate a group of polyacylated anthocyanins, named ternatins, in their petals. The first step in ternatin biosynthesis is the transfer of glucose from UDP-glucose to anthocyanidins such as delphinidin, a reaction catalyzed in C. ternatea by UDP-glucose:anthocyanidin 3-O-glucosyltransferase (Ct3GT-A; AB185904). To elucidate the structure-function relationship of Ct3GT-A, recombinant Ct3GT-A was expressed in Escherichia coli and its tertiary structure was determined to 1.85 Å resolution by using X-ray crystallography. The structure of Ct3GT-A shows a common folding topology, the GT-B fold, comprised of two Rossmann-like ß/α/ß domains and a cleft located between the N- and C-domains containing two cavities that are used as binding sites for the donor (UDP-Glc) and acceptor substrates. By comparing the structure of Ct3GT-A with that of the flavonoid glycosyltransferase VvGT1 from red grape (Vitis vinifera) in complex with UDP-2-deoxy-2-fluoro glucose and kaempferol, locations of the catalytic His-Asp dyad and the residues involved in recognizing UDP-2-deoxy-2-fluoro glucose were essentially identical in Ct3GT-A, but certain residues of VvGT1 involved in binding kaempferol were found to be substituted in Ct3GT-A. These findings are important for understanding the differentiation of acceptor-substrate recognition in these two enzymes.
Subject(s)
Anthocyanins/chemistry , Clitoria/enzymology , Glucosyltransferases/chemistry , Uridine Diphosphate Glucose/chemistry , Base Sequence , Crystallography, X-Ray , DNA Primers , Molecular Sequence Data , Polymerase Chain ReactionABSTRACT
Assembly of the eukaryotic 20S proteasome is an ordered process involving several proteins operating as proteasome assembly factors including PAC1-PAC2 but archaeal 20S proteasome subunits can spontaneously assemble into an active cylindrical architecture. Recent bioinformatic analysis identified archaeal PAC1-PAC2 homologs PbaA and PbaB. However, it remains unclear whether such assembly factor-like proteins play an indispensable role in orchestration of proteasome subunits in archaea. We revealed that PbaB forms a homotetramer and exerts a dual function as an ATP-independent proteasome activator and a molecular chaperone through its tentacle-like C-terminal segments. Our findings provide insights into molecular evolution relationships between proteasome activators and assembly factors.
Subject(s)
Archaea/metabolism , Archaeal Proteins/metabolism , Proteasome Endopeptidase Complex/metabolism , Evolution, Molecular , Protein BindingABSTRACT
HOIL-1L and its binding partner HOIP are essential components of the E3-ligase complex that generates linear ubiquitin (Ub) chains, which are critical regulators of NF-κB activation. Using crystallographic and mutational approaches, we characterize the unexpected structural basis for the specific interaction between the Ub-like domain (UBL) of HOIL-1L and the Ub-associated domain (UBA) of HOIP. Our data indicate the functional significance of this non-canonical mode of UBA-UBL interaction in E3 complex formation and subsequent NF-κB activation. This study highlights the versatility and specificity of protein-protein interactions involving Ub/UBLs and their cognate proteins.
Subject(s)
Ubiquitin-Protein Ligases/chemistry , Ubiquitin-Protein Ligases/metabolism , Ubiquitin/chemistry , Ubiquitin/metabolism , Carrier Proteins/chemistry , Carrier Proteins/metabolism , Cell Line , Circular Dichroism , Humans , Immunoprecipitation , Magnetic Resonance Spectroscopy , NF-kappa B/metabolism , Protein Binding , Protein Structure, Secondary , Surface Plasmon Resonance , Transcription Factors , UltracentrifugationABSTRACT
Proteasomal degradation is mediated through modification of target proteins by Lys-48-linked polyubiquitin (polyUb) chain, which interacts with several binding partners in this pathway through hydrophobic surfaces on individual Ub units. However, the previously reported crystal structures of Lys-48-linked diUb exhibit a closed conformation with sequestered hydrophobic surfaces. NMR studies on mutated Lys-48-linked diUb indicated a pH-dependent conformational equilibrium between closed and open states with the predominance of the former under neutral conditions (90% at pH 6.8). To address the question of how Ub-binding proteins can efficiently access the sequestered hydrophobic surfaces of Ub chains, we revisited the conformational dynamics of Lys-48-linked diUb in solution using wild-type diUb and cyclic forms of diUb in which the Ub units are connected through two Lys-48-mediated isopeptide bonds. Our newly determined crystal structure of wild-type diUb showed an open conformation, whereas NMR analyses of cyclic Lys-48-linked diUb in solution revealed that its structure resembled the closed conformation observed in previous crystal structures. Comparison of a chemical shift of wild-type diUb with that of monomeric Ub and cyclic diUb, which mimic the open and closed states, respectively, with regard to the exposure of hydrophobic surfaces to the solvent indicates that wild-type Lys-48-linked diUb in solution predominantly exhibits the open conformation (75% at pH 7.0), which becomes more populated upon lowering pH. The intrinsic properties of Lys-48-linked Ub chains to adopt the open conformation may be advantageous for interacting with Ub-binding proteins.
Subject(s)
Protein Multimerization/physiology , Ubiquitin/chemistry , Crystallography, X-Ray , Humans , Hydrophobic and Hydrophilic Interactions , Nuclear Magnetic Resonance, Biomolecular , Protein Structure, Quaternary , Ubiquitin/metabolismABSTRACT
Most methanogenic archaea reduce CO(2) with H(2) to CH(4). For the activation of H(2), they use different [NiFe]-hydrogenases, namely energy-converting [NiFe]-hydrogenases, heterodisulfide reductase-associated [NiFe]-hydrogenase or methanophenazine-reducing [NiFe]-hydrogenase, and F(420)-reducing [NiFe]-hydrogenase. The energy-converting [NiFe]-hydrogenases are phylogenetically related to complex I of the respiratory chain. Under conditions of nickel limitation, some methanogens synthesize a nickel-independent [Fe]-hydrogenase (instead of F(420)-reducing [NiFe]-hydrogenase) and by that reduce their nickel requirement. The [Fe]-hydrogenase harbors a unique iron-guanylylpyridinol cofactor (FeGP cofactor), in which a low-spin iron is ligated by two CO, one C(O)CH(2)-, one S-CH(2)-, and a sp(2)-hybridized pyridinol nitrogen. Ligation of the iron is thus similar to that of the low-spin iron in the binuclear active-site metal center of [NiFe]- and [FeFe]-hydrogenases. Putative genes for the synthesis of the FeGP cofactor have been identified. The formation of methane from 4 H(2) and CO(2) catalyzed by methanogenic archaea is being discussed as an efficient means to store H(2).
Subject(s)
Archaea/enzymology , Hydrogen/metabolism , Hydrogenase/metabolism , Nickel , Archaea/metabolism , Hydrogenase/chemistry , Hydrogenase/geneticsABSTRACT
The Tk-idsB encoding cis-prenyltransferase which catalyzes consecutive cis-condensation of isopentenyl diphosphate to allylic diphosphate was isolated from a hyperthermophilic archaeon Thermococcus kodakaraensis, and enzymatic characteristics of the recombinant Tk-IdsB were examined. Tk-IdsB was not fully denatured even at 90 degrees C and preferably utilizes both C(10) and C(15) allylic diphosphates to yield mainly the C(60)-C(65) products. Based on structural models, single alanine-substitution mutants at Glu68, Lys109, or Leu113 were constructed, showing that all the three produced longer chains (C(65)-C(70)) than the wild-type and the substitution at 109 (K109A) was the most effective. Tk-IdsB was applied to an organic-aqueous dual-phase system and more than 90% of the products were recovered from the organic phase when 1-butanol or 1-pentanol was overlaid. When 1-octanol was overlaid, 70% of the products were obtained from the upper organic phase. The product distributions were changed depending on the hydrophobicity of organic solvents used. Tk-IdsB was then immobilized onto silica beads to make Tk-IdsB more tolerant, showing that half-life of enzyme at 80 degrees C was prolonged by immobilization. When the immobilized Tk-IdsB was applied in the organic-aqueous dual-phase system, immobilized Tk-IdsB catalyzed consecutive condensation more efficiently than the unimmobilized one.
Subject(s)
Enzymes, Immobilized/metabolism , Hemiterpenes/metabolism , Organophosphorus Compounds/metabolism , Thermococcus/enzymology , Transferases/metabolism , 1-Octanol/chemistry , Enzyme Stability , Enzymes, Immobilized/chemistry , Enzymes, Immobilized/genetics , Kinetics , Models, Molecular , Mutagenesis, Site-Directed , Thermococcus/genetics , Transferases/chemistry , Transferases/geneticsABSTRACT
[Fe]-hydrogenase is one of three types of enzymes known to activate H(2). Crystal structure analysis recently revealed that its active site iron is ligated square-pyramidally by Cys176-sulfur, two CO, an "unknown" ligand and the sp(2)-hybridized nitrogen of a unique iron-guanylylpyridinol-cofactor. We report here on the structure of the C176A mutated enzyme crystallized in the presence of dithiothreitol (DTT). It suggests an iron center octahedrally coordinated by one DTT-sulfur and one DTT-oxygen, two CO, the 2-pyridinol's nitrogen and the 2-pyridinol's 6-formylmethyl group in an acyl-iron ligation. This result led to a re-interpretation of the iron ligation in the wild-type.
Subject(s)
Hydrogenase/chemistry , Hydrogenase/metabolism , Iron-Sulfur Proteins/chemistry , Iron-Sulfur Proteins/metabolism , Iron/chemistry , Iron/metabolism , Adenine/metabolism , Catalytic Domain , Crystallography, X-Ray , Cytosine/metabolism , Holoenzymes/chemistry , Holoenzymes/genetics , Holoenzymes/metabolism , Hydrogenase/genetics , Iron-Sulfur Proteins/genetics , Methanococcales/enzymology , Methanococcales/genetics , Mutation/genetics , Protein Structure, QuaternaryABSTRACT
MobR from Comamonas testosteroni KH122-3s is a member of the MarR family of transcriptional regulators and functions as a repressor for the mobA gene that encodes a 3-hydroxybenzoate 4-hydroxylase. 3-Hydroxybenzoate binds to MobR as a ligand, resulting in an efficient induction of mobA. Various 3-hydroxybenzoate analogues were examined for their inducibilities using the mobA::lacZ transcriptional fusion system. beta-Galactosidase was induced by the addition of 2,3-dihydroxybenzoate or 3,5-dihydroxybenzoate besides 3-hydroxybenzoate, suggesting that the hydroxyl group at position 3 is critical in addition to the carboxyl group on the aromatic ring. A gel mobility-shift assay also showed that MobR was released from the target DNA in the presence of these compounds. Circular dichroism studies demonstrated that MobR adopted two conformational states corresponding to the 3-hydroxybenzoate-bound and unbound forms. Other ligands also induced the structural change as well; however, the tertiary structures of converted forms were different from those by 3-hydroxybenzoate.
Subject(s)
Bacterial Proteins/metabolism , Comamonas testosteroni/metabolism , DNA-Binding Proteins/metabolism , Mixed Function Oxygenases/metabolism , Bacterial Proteins/genetics , Circular Dichroism , Comamonas testosteroni/genetics , DNA-Binding Proteins/genetics , Hydroxybenzoates/chemistry , Hydroxybenzoates/metabolism , Hydroxybenzoates/pharmacology , Kinetics , Ligands , Mixed Function Oxygenases/genetics , Molecular Structure , Phenol/chemistry , Phenol/metabolism , Phenol/pharmacology , Protein Binding/drug effects , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , ResorcinolsABSTRACT
The 3-hydroxybenzoate hydroxylase (MHBH) from Comamonas testosteroni KH122-3s is a single-component flavoprotein monooxygenase, a member of the glutathione reductase (GR) family. It catalyzes the conversion of 3-hydroxybenzoate to 3,4-dihydroxybenzoate with concomitant requirements for equimolar amounts of NADPH and molecular oxygen. The production of dihydroxy-benzenoid derivative by hydroxylation is the first step in the aerobic degradation of various phenolic compounds in soil microorganisms. To establish the structural basis for substrate recognition, the crystal structure of MHBH in complex with its substrate was determined at 1.8 A resolution. The enzyme is shown to form a physiologically active homodimer with crystallographic 2-fold symmetry, in which each subunit consists of the first two domains comprising an active site and the C-terminal domain involved in oligomerization. The protein fold of the catalytic domains and the active-site architecture, including the FAD and substrate-binding sites, are similar to those of 4-hydroxybenzoate hydroxylase (PHBH) and phenol hydroxylase (PHHY), which are members of the GR family, providing evidence that the flavoprotein aromatic hydroxylases share similar catalytic actions for hydroxylation of the respective substrates. Structural comparison of MHBH with the homologous enzymes suggested that a large tunnel connecting the substrate-binding pocket to the protein surface serves for substrate transport in this enzyme. The internal space of the large tunnel is distinctly divided into hydrophilic and hydrophobic regions. The characteristically stratified environment in the tunnel interior and the size of the entrance would allow the enzyme to select its substrate by amphiphilic nature and molecular size. In addition, the structure of the Xe-derivative at 2.5 A resolution led to the identification of a putative oxygen-binding site adjacent to the substrate-binding pocket. The hydrophobic nature of the xenon-binding site extends to the solvent through the tunnel, suggesting that the tunnel could be involved in oxygen transport.