Crystal structure of the terminal oxygenase component of biphenyl dioxygenase derived from Rhodococcus sp. strain RHA1

Biphenyl dioxygenase is the enzyme that catalyzes the stereospecific dioxygenation of the aromatic ring. This enzyme has attracted the attention of researchers due to its ability to oxidize polychlorinated biphenyls, which is one of the serious environmental contaminants. We determined the crystal structure of the terminal oxygenase component of the biphenyl dioxygenase (BphA1A2) derived from Rhodococcus strain sp. RHA1 in substrate-free and complex forms. These crystal structures revealed that the substrate-binding pocket makes significant conformational changes upon substrate binding to accommodate the substrate into the pocket. Our analysis of the crystal structures suggested that the residues in the substrate-binding pocket can be classified into three groups, which, respectively, seem to be responsible for the catalytic reaction, the orientation/conformation of the substrate, and the conformational changes of the substrate-binding pocket. The cooperative actions of residues in the three groups seem to determine the substrate specificity of the enzyme.     

Structural prototypes for an extended family of flavoprotein reductases: Comparison of phthalate dioxygenase reductase with ferredoxin reductase and ferredoxin

The structure of phthalate dioxygenase reductase (PDR), a monomeric iron-sulfur flavoprotein that delivers electrons from NADH to phthalate dioxygenase, is compared to ferredoxin-NADP⁺ reductase (FNR) and ferredoxin, the proteins that reduce NADP⁺ in the final reaction of photosystem I. The folding patterns of the domains that bind flavin, NAD(P), and [2Fe-2S] are very similar in the two systems. Alignment of the X-ray structures of PDR and FNR substantiates the assignment of features that characterize a family of flavoprotein reductases whose members include cytochrome P-450 reductase, sulfite and nitrate reductases, and nitric oxide synthase. Hallmarks of this subfamily of flavoproteins, here termed the FNR family, are an antiparallel β-barrel that binds the flavin prosthetic group, and a characteristic variant of the classic pyridine nucleotide-binding fold. Despite the similarities between FNR and PDR, attempts to model the structure of a dissociable FNR:ferredoxin complex by analogy with PDR reveal features that are at odds with chemical crosslinking studies (Zanetti, G., Morelli, D., Ronchi, S., Negri, A., Aliverti, A., & Curti, B., 1988, Biochemistry 27, 3753–3759). Differences in the binding sites for flavin and pyridine nucleotides determine the nucleotide specificities of FNR and PDR. The specificity of FNR for NADP⁺ arises primarily from substitutions in FNR that favor interactions with the 2′ phosphate of NADP⁺. Variations in the conformation and sequences of the loop adjoining the flavin phosphate affect the selectivity for FAD versus FMN. The midpoint potentials for reduction of the flavin and [2Fe–2S] groups in PDR are higher than their counterparts in FNR and spinach ferredoxin, by about 120 mV and 260 mV, respectively. Comparisons of the structure of PDR with spinach FNR and with ferredoxin from Anabaena 7120, along with calculations of electrostatic potentials, suggest that local interactions, including hydrogen bonds, are the dominant contributors to these differences in potential.     

Molecular mechanism of the redox-dependent interaction between NADH-dependent ferredoxin reductase and Rieske-type [2Fe-2S] ferredoxin

The electron transfer system of the biphenyl dioxygenase BphA, which is derived from Acidovorax sp. (formally Pseudomonas sp.) strain KKS102, is composed of an FAD-containing NADH-ferredoxin reductase (BphA4) and a Rieske-type [2Fe-2S] ferredoxin (BphA3). Biochemical studies have suggested that the whole electron transfer process from NADH to BphA3 comprises three consecutive elementary electron-transfer reactions, in which BphA3 and BphA4 interact transiently in a redox-dependent manner. Initially, BphA4 receives two electrons from NADH. The reduced BphA4 then delivers one electron each to the [2Fe-2S] cluster of the two BphA3 molecules through redox-dependent transient interactions. The reduced BphA3 transports the electron to BphA1A2, a terminal oxygenase, to support the activation of dioxygen for biphenyl dihydroxylation. In order to elucidate the molecular mechanisms of the sequential reaction and the redox-dependent interaction between BphA3 and BphA4, we determined the crystal structures of the productive BphA3-BphA4 complex, and of free BphA3 and BphA4 in all the redox states occurring in the catalytic cycle. The crystal structures of these reaction intermediates demonstrated that each elementary electron transfer induces a series of redox-dependent conformational changes in BphA3 and BphA4, which regulate the interaction between them. In addition, the conformational changes induced by the preceding electron transfer seem to induce the next electron transfer. The interplay of electron transfer and induced conformational changes seems to be critical to the sequential electron-transfer reaction from NADH to BphA3.     

A cluster exposed: structure of the Rieske ferredoxin from biphenyl dioxygenase and the redox properties of Rieske Fe-S proteins

BACKGROUND: Ring-hydroxylating dioxygenases are multicomponent systems that initiate biodegradation of aromatic compounds. Many dioxygenase systems include Rieske-type ferredoxins with amino acid sequences and redox properties remarkably different from the Rieske proteins of proton-translocating respiratory and photosynthetic complexes. In the latter, the [Fe2S2] clusters lie near the protein surface, operate at potentials above +300 mV at pH 7, and express pH- and ionic strength-dependent redox behavior. The reduction potentials of the dioxygenase ferredoxins are approximately 150 mV and are pH-independent. These distinctions were predicted to arise from differences in the exposure of the cluster and/or interactions of the histidine ligands. RESULTS: The crystal structure of BphF, the Rieske-type ferredoxin associated with biphenyl dioxygenase, was determined by multiwavelength anomalous diffraction and refined at 1.6 A resolution. The structure of BphF was compared with other Rieske proteins at several levels. BphF has the same two-domain fold as other Rieske proteins, but it lacks all insertions that give the others unique structural features. The BphF Fe-S cluster and its histidine ligands are exposed. However, the cluster has a significantly different environment in that five fewer polar groups interact strongly with the cluster sulfide or the cysteinyl ligands. CONCLUSIONS: BphF has structural features consistent with a minimal and perhaps archetypical Rieske protein. Variations in redox potentials among Rieske clusters appear to be largely the result of local electrostatic interactions with protein partial charges. Moreover, it appears that the redox-linked ionizations of the Rieske proteins from proton-translocating complexes are also promoted by these electrostatic interactions.     

Crystal structure of human homogentisate dioxygenase

Homogentisate dioxygenase (HGO) cleaves the aromatic ring during the metabolic degradation of Phe and Tyr. HGO deficiency causes alkaptonuria (AKU), the first human disease shown to be inherited as a recessive Mendelian trait. Crystal structures of apo-HGO and HGO containing an iron ion have been determined at 1.9 and 2.3 A resolution, respectively. The HGO protomer, which contains a 280-residue N-terminal domain and a 140-residue C-terminal domain, associates as a hexamer arranged as a dimer of trimers. The active site iron ion is coordinated near the interface between subunits in the HGO trimer by a Glu and two His side chains. HGO represents a new structural class of dioxygenases. The largest group of AKU associated missense mutations affect residues located in regions of contact between subunits.     

Crystal structure of the terminal oxygenase component of cumene dioxygenase from Pseudomonas fluorescens IP01

The crystal structure of the terminal component of the cumene dioxygenase multicomponent enzyme system of Pseudomonas fluorescens IP01 (CumDO) was determined at a resolution of 2.2 A by means of molecular replacement by using the crystal structure of the terminal oxygenase component of naphthalene dioxygenase from Pseudomonas sp. strain NCIB 9816-4 (NphDO). The ligation of the two catalytic centers of CumDO (i.e., the nonheme iron and Rieske [2Fe-2S] centers) and the bridging between them in neighboring catalytic subunits by hydrogen bonds through a single amino acid residue, Asp231, are similar to those of NphDO. An unidentified external ligand, possibly dioxygen, was bound at the active site nonheme iron. The entrance to the active site of CumDO is different from the entrance to the active site of NphDO, as the two loops forming the lid exhibit great deviation. On the basis of the complex structure of NphDO, a biphenyl substrate was modeled in the substrate-binding pocket of CumDO. The residues surrounding the modeled biphenyl molecule include residues that have already been shown to be important for its substrate specificity by a number of engineering studies of biphenyl dioxygenases.     

Crystal structure of a ferredoxin reductase for the CYP199A2 system from Rhodopseudomonas palustris

Cytochrome P450‐199A2 from Rhodopseudomonas palustris oxidizes para‐substituted benzoic acids and may play a role in lignin and aromatic acid degradation pathways in the bacterium. CYP199A2 has an associated [2Fe‐2S] ferredoxin, palustrisredoxin (Pux) but not a ferredoxin reductase. A genome search identified the palustrisredoxin reductase (PuR) gene. PuR was produced in Escherichia coli and shown to be a flavin‐dependent protein that supports efficient electron transfer from NADH to Pux, thus reconstituting CYP199A2 monooxygenase activity (k_cat = 37.9 s^–1 with 4‐methoxybenzoic acid). The reduction of Pux by PuR shows K_m = 4.2 μM and k_cat = 262 s^–1 in 50 mM Tris, pH 7.4. K_m is increased to 154 μM in the presence of 200 mM KCl, indicating the importance of ionic interactions in PuR/Pux binding. The crystal structure of PuR has been determined at 2.2 Å resolution and found to be closely related to that of other oxygenase‐coupled NADH‐dependent ferredoxin reductases. Residues on the surface that had been proposed to be involved in ferredoxin reductase‐ferredoxin binding are conserved in PuR. However, Lys328 in PuR lies over the FAD isoalloxazine ring and, together with His11 and Gln41, render the electrostatic potential of the surface more positive and may account for the greater involvement of electrostatic interactions in ferredoxin binding by PuR. Consistent with these observations the K328G mutation weakened Pux binding and virtually eliminated the dependence of PuR/Pux binding on salt concentration, thus confirming that the FAD si side surface in the vicinity of Lys328 is the ferredoxin binding site. Proteins 2009. © 2009 Wiley‐Liss, Inc.     

Crystal structure of rat biliverdin reductase

Biliverdin reductase (BVR) is a soluble cytoplasmic enzyme that catalyzes the conversion of biliverdin to bilirubin using NADH or NADPH as electron donor. Bilirubin is a significant biological antioxidant, but it is also neurotoxic and the cause of kernicterus. In this study, we have determined the crystal structure of rat BVR at 1.4 A resolution. The structure contains two domains: an N-terminal domain characteristic of a dinucleotide binding fold (Rossmann fold) and a C-terminal domain that is predominantly an antiparallel six-stranded beta-sheet. Based on this structure, we propose modes of binding for NAD(P)H and biliverdin, and a possible mechanism for the enzyme.     

Identification of the bphA4 gene encoding ferredoxin reductase involved in biphenyl and polychlorinated biphenyl degradation in Pseudomonas sp. strain KKS102.

The nucleotide sequence of the downstream region of the bph operon from Pseudomonas sp. strain KKS102 was determined. Two open reading frames (ORF1 and ORF2) were found in this region, and the deduced amino acid sequence of ORF2 showed homology with the sequences of four ferredoxin reductases of dioxygenase systems. When this region was inserted just upstream of the bph operon, which does not contain a gene encoding ferredoxin reductase, biphenyl dioxygenase activity was detected. The 24- and 44-kDa polypeptides predicted from the two open reading frames were identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Crude extract which contained the products of ORF2 and bphA1A2A3 showed cytochrome c reduction activity. These data clearly suggest that ORF2 encodes ferredoxin reductase. The deduced amino acid sequence of ORF1 does not show significant homology with the sequences of any other proteins in the SWISS-PROT data bank, and the function of ORF1 is unknown.     

Crystal structure of the non-heme iron dioxygenase PtlH in pentalenolactone biosynthesis

The non-heme iron dioxygenase PtlH from the soil organism Streptomyces avermitilis is a member of the iron(II)/alpha-ketoglutarate-dependent dioxygenase superfamily and catalyzes an essential reaction in the biosynthesis of the sesquiterpenoid antibiotic pentalenolactone. To investigate the structural basis for substrate recognition and catalysis, we have determined the x-ray crystal structure of PtlH in several complexes with the cofactors iron, alpha-ketoglutarate, and the non-reactive enantiomer of the substrate, ent-1-deoxypentalenic acid, in four different crystal forms to up to 1.31 A resolution. The overall structure of PtlH forms a double-stranded barrel helix fold, and the cofactor-binding site for iron and alpha-ketoglutarate is similar to other double-stranded barrel helix fold enzymes. Additional secondary structure elements that contribute to the substrate-binding site in PtlH are not conserved in other double-stranded barrel helix fold enzymes. Binding of the substrate enantiomer induces a reorganization of the monoclinic crystal lattice leading to a disorder-order transition of a C-terminal alpha-helix. The newly formed helix blocks the major access to the active site and effectively traps the bound substrate. Kinetic analysis of wild type and site-directed mutant proteins confirms a critical function of two arginine residues in substrate binding, while simulated docking of the enzymatic reaction product reveals the likely orientation of bound substrate.     

Crystal structure of the catalytic core component of the alkylhydroperoxide reductase AhpF from Escherichia coli

Alkylhydroperoxide reductases (AhpR, EC 1.6.4.*) are essential for the oxygen tolerance of aerobic organisms by converting otherwise toxic hydroperoxides of lipids or nucleic acids to the corresponding alcohols. The AhpF component belongs to the family of pyridine nucleotide-disulphide oxidoreductases and channels electrons from NAD(P)H towards the AhpC component which finally reduces cognate substrates. The structure of the catalytic core of the Escherichia coli AhpF (A212-A521) with a bound FAD cofactor was determined at 1.9 A resolution in its oxidized state. The dimeric arrangement of the AhpF catalytic core and the predicted interaction mode between the N-terminal PDO-like domain and the NADPH domain favours an intramolecular electron transfer between the two redox-active disulphide centres of AhpF.     

Alteration of regiospecificity in biphenyl dioxygenase by active-site engineering

Biphenyl dioxygenase (Bph Dox) is responsible for the initial dioxygenation step during the metabolism of biphenyl. The large subunit (BphA1) of Bph Dox plays a crucial role in the determination of the substrate specificity of biphenyl-related compounds, including polychlorinated biphenyls (PCBs). Based on crystallographic analyses of naphthalene dioxygenase (B. Kauppi, K. Lee, E. Carredano, R. E. Parales, D. T. Gibson, H. Eklund, and S. Ramaswamy, Structure 6:571-586, 1998), we developed a three-dimensional model of KF707 BphA1 of Pseudomonas pseudoalcaligenes KF707. Based on structural information about the amino acids which coordinate the catalytic nonheme iron center, we constructed 12 site-directed BphA1 mutants with changes at positions 227, 332, 335, 376, 377, and 383 and expressed these enzymes in Escherichia coli. The Ile335Phe, Thr376Asn, and Phe377Leu Bph Dox mutants exhibited altered regiospecificities for various PCBs compared with wild-type Bph Dox. In particular, the Ile335Phe mutant acquired the ability to degrade 2,5,2',5'-tetrachlorobiphenyl by 3,4-dioxygenation and showed bifunctional 2,3-dioxygenase and 3,4-dioxygenase activities for 2,5,2'-trichlorobiphenyl and 2,5,4'-trichlorobiphenyl. Furthermore, two mutants, the Phe227Val and Phe377Ala mutants, introduced molecular oxygen at the 2,3 position, forming 3-chloro-2',3'-dihydroxy biphenyl with concomitant dechlorination.     

NADH-dependent cinerulose reductase in rat liver microsomes.

In the course of studies on the metabolism of a new antitumor anthracycline antibiotic, aclacinomycin A, the new keto reductase which catalyzes the reduction of keto group of L-cinerulose of aclacinomycin A to L-rhodinose was found in rat liver microsomal membrane. The enzyme requires NADH for the reduction and showed optimum pH at 7.0. Km value for aclacinomycin A, 2.1 × 10^?5 M and the concentration of NADH need to half maximal activity, 6.2 × 10^?5 M were obtained. The activity was potently inhibited by detergents, such as Triton X-100, sodium deoxycholate and sodium dodecyl sulfate.     

Crystallization of the terminal oxygenase component of biphenyl dioxygenase derived from Rhodococcus sp. strain RHA1

The terminal oxygenase component of the biphenyl dioxygenase (BphA1A2 complex) was over-expressed with a novel over expression system in recombinant Rhodococcus strain and purified. The purified enzyme has been crystallized by the hanging drop vapor diffusion method and subjected to X-ray diffraction analysis. The crystals belong to the tetragonal system in the space group P4(1)2(1)2 or P4(3)2(1)2 and diffract to better than 2.2A resolution.     

The structure and biochemistry of NADH-dependent cytochrome b5 reductase are now consistent.

Cytochrome b5 reductase (cb5r) (EC 1.6.6.2) catalyzes the reduction of two molecules of cytochrome b5 using NADH as the physiological electron donor. The structure of pig cb5r at 2.4 A resolution was previously reported in the literature, but it was inconsistent with the biochemistry; for example, K83 and C245 were both implicated in the mechanism, but were not located at the active site. To address this problem, we have determined the structures of cb5r from rat at 2.0 A resolution and in a complex with NAD+ at 2.3 A resolution. We found significant differences throughout the rat structure compared to that of pig, including the locations of the lysine and cysteine residues mentioned above. To test the structural models, we made single amino acid substitutions of this lysine and showed that all substitutions produced correctly folded proteins and exhibited normal flavin behavior. However, the apparent kcat(NADH) decreased, and the apparent K(m) for NADH increased; the K(m)'s for cytochrome b5 were unchanged relative to that of the wild type. The largest effect was for the glutamate-substituted protein, which was further characterized using a charge transfer assay and found to be less efficient at NADH utilization than the wild type. These results are consistent with a role for this lysine in stabilizing the NADH-bound form of cb5r. We have concluded that the pig structure was mistraced in several regions and have reinterpreted mutants in these regions that give rise to the hereditary disease methemoglobinemia.     

Characterization and X-ray structure of the NADH-dependent coenzyme A disulfide reductase from Thermus thermophilus

The crystal structure of the enzyme previously characterized as a type-2 NADH:menaquinone oxidoreductase (NDH-2) from Thermus thermophilus has been solved at a resolution of 2.9 Å and revealed that this protein is, in fact, a coenzyme A-disulfide reductase (CoADR). Coenzyme A (CoASH) replaces glutathione as the major low molecular weight thiol in Thermus thermophilus and is maintained in the reduced state by this enzyme (CoADR). Although the enzyme does exhibit NADH:menadione oxidoreductase activity expected for NDH-2 enzymes, the specific activity with CoAD as an electron acceptor is about 5-fold higher than with menadione. Furthermore, the crystal structure contains coenzyme A covalently linked Cys44, a catalytic intermediate (Cys44-S-S-CoA) reduced by NADH via the FAD cofactor. Soaking the crystals with menadione shows that menadione can bind to a site near the redox active FAD, consistent with the observed NADH:menadione oxidoreductase activity. CoADRs from other species were also examined and shown to have measurable NADH:menadione oxidoreductase activity. Although a common feature of this family of enzymes, no biological relevance is proposed. The CoADR from T. thermophilus is a soluble homodimeric enzyme. Expression of the recombinant TtCoADR at high levels in E. coli results in a small fraction that co-purifies with the membrane fraction, which was used previously to isolate the enzyme wrongly identified as a membrane-bound NDH-2. It is concluded that T. thermophilus does not contain an authentic NDH-2 component in its aerobic respiratory chain.     

The crystal structure of NADPH:ferredoxin reductase from Azotobacter vinelandii.

NADPH:ferredoxin reductase (AvFPR) is involved in the response to oxidative stress in Azotobacter vinelandii. The crystal structure of AvFPR has been determined at 2.0 A resolution. The polypeptide fold is homologous with six other oxidoreductases whose structures have been solved including Escherichia coli flavodoxin reductase (EcFldR) and spinach, and Anabaena ferredoxin:NADP+ reductases (FNR). AvFPR is overall most homologous to EcFldR. The structure is comprised of a N-terminal six-stranded antiparallel beta-barrel domain, which binds FAD, and a C-terminal five-stranded parallel beta-sheet domain, which binds NADPH/NADP+ and has a classical nucleotide binding fold. The two domains associate to form a deep cleft where the NADPH and FAD binding sites are juxtaposed. The structure displays sequence conserved motifs in the region surrounding the two dinucleotide binding sites, which are characteristic of the homologous enzymes. The folded over conformation of FAD in AvFPR is similar to that in EcFldR due to stacking of Phe255 on the adenine ring of FAD, but it differs from that in the FNR enzymes, which lack a homologous aromatic residue. The structure of AvFPR displays three unique features in the environment of the bound FAD. Two features may affect the rate of reduction of FAD: the absence of an aromatic residue stacked on the isoalloxazine ring in the NADPH binding site; and the interaction of a carbonyl group with N10 of the flavin. Both of these features are due to the substitution of a conserved C-terminal tyrosine residue with alanine (Ala254) in AvFPR. An additional unique feature may affect the interaction of AvFPR with its redox partner ferredoxin I (FdI). This is the extension of the C-terminus by three residues relative to EcFldR and by four residues relative to FNR. The C-terminal residue, Lys258, interacts with the AMP phosphate of FAD. Consequently, both phosphate groups are paired with a basic group due to the simultaneous interaction of the FMN phosphate with Arg51 in a conserved FAD binding motif. The fourth feature, common to homologous oxidoreductases, is a concentration of 10 basic residues on the face of the protein surrounding the active site, in addition to Arg51 and Lys258.     

Interaction of ferredoxin with ferredoxin:NADP reductase: effects of chemical modification of ferredoxin

Chemical modification studies have been conducted on spinach ferredoxin to determine the nature of the groups on ferredoxin involved in its interaction with its reaction partners. Modification of a limited number (three or four) carboxyl groups or of the single histidine residue resulted in a decreased ability of ferredoxin to participate in NADP photoreduction but not in cytochrome c photoreduction, suggesting that these groups may be involved in interaction with ferredoxin:NADP reductase but are not involved in interaction with the reducing side of Photosystem I. In contrast, modification of amino groups or the single arginine residue on ferredoxin had little effect on the ability of ferredoxin to participate in NADP photoreduction, suggesting these groups are not involved in the interaction of ferredoxin with either ferredoxin:NADP reductase or the reducing side of Photosystem I. Attempts to modify tyrosine residues on ferredoxin resulted in destruction of the iron-sulfur center of the protein.     

Properties of ferredoxin reductase and ferredoxin from the bovine corpus luteum

• 1.1. Ferredoxin reductase and ferredoxin were purified from the bovine corpus luteum and their properties compared to the corresponding adrenal proteins.
• 2.2. The luteal and adrenal proteins had similar absorbance spectra and molecular weights.
• 3.3. Evidence was obtained from spectrophotometric titrations for formation of 1:1 complexes between luteal ferredoxin reductase and ferredoxin and between ferredoxin and cytochrome P-450_scc.
• 4.4. Adrenal ferredoxin reductase and ferredoxin were equally as effective as luteal ferredoxin reductase and ferredoxin in supporting cholesterol side-chain cleavage by luteal cytochrome P-450_scc.

     

Crystal structure of human pyrroline-5-carboxylate reductase

Pyrroline-5-carboxylate reductase (P5CR) is a universal housekeeping enzyme that catalyzes the reduction of Delta(1)-pyrroline-5-carboxylate (P5C) to proline using NAD(P)H as the cofactor. The enzymatic cycle between P5C and proline is very important for the regulation of amino acid metabolism, intracellular redox potential, and apoptosis. Here, we present the 2.8 Angstroms resolution structure of the P5CR apo enzyme, its 3.1 Angstroms resolution ternary complex with NAD(P)H and substrate-analog. The refined structures demonstrate a decameric architecture with five homodimer subunits and ten catalytic sites arranged around a peripheral circular groove. Mutagenesis and kinetic studies reveal the pivotal roles of the dinucleotide-binding Rossmann motif and residue Glu221 in the human enzyme. Human P5CR is thermostable and the crystals were grown at 37 degrees C. The enzyme is implicated in oxidation of the anti-tumor drug thioproline.