Identification and Characterization of an OSH1 Thiol Reductase from Populus Trichocarpa.

Interferon gamma-induced lysosomal thiol reductase (GILT) is abundantly expressed in antigen-presenting cells and participates in the treatment and presentation of antigens by major histocompatibility complex II. Also, GILT catalyzes the reduction of disulfide bonds, which plays an important role in cellular immunity. (1) Background: At present, the studies of GILT have mainly focused on animals. In plants, GILT homologous gene (Arabidopsis thalianaOSH1: AtOSH1) was discovered in the forward screen of mutants with compromised responses to sulphur nutrition. However, the complete properties and functions of poplar OSH1 are unclear. In addition, CdCl2 stress is swiftly engulfing the limited land resources on which humans depend, restricting agricultural production. (2) Methods: A prokaryotic expression system was used to produce recombinant PtOSH1 protein, and Western blotting was performed to identify its activity. In addition, a simplified version of the floral-dip method was used to transform A. thaliana. (3) Results: Here, we describe the identification and characterization of OSH1 from Populus trichocarpa. The deduced PtOSH1 sequence contained CQHGX2ECX2NX4C and CXXC motifs. The transcript level of PtOSH1 was increased by cadmium (Cd) treatment. In addition, recombinant PtOSH1 reduced disulfide bonds. A stress assay showed that PtOSH1-overexpressing (OE) A. thaliana lines had greater resistance to Cd than wild-type (WT) plants. Also, the activities of superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT) in PtOSH1-OE plants were significantly higher than those in WT A. thaliana. These results indicate that PtOSH1 likely plays an important role in the response to Cd by regulating the reactive oxygen species (ROS)-scavenging system. (4) Conclusions: PtOSH1 catalyzes the reduction of disulfide bonds and behaves as a sulfhydryl reductase under acidic conditions. The overexpression of PtOSH1 in A. thaliana promoted root development, fresh weight, and dry weight; upregulated the expression levels of ROS scavenging-related genes; and improved the activity of antioxidant enzymes, enhancing plant tolerance to cadmium (Cd) stress. This study aimed to provide guidance that will facilitate future studies of the function of PtOSH1 in the response of plants to Cd stress.

Analysis of some enzymes activities of hydrogen sulfide metabolism in plants.

Hydrogen sulfide (H2S) which is considered as a novel gasotransmitter after reactive oxygen species and nitric oxide in plants has dual character, that is, toxicity that inhibits cytochrome oxidase at high concentration and as signal molecule which is involved in plant growth, development, and the acquisition of tolerance to adverse environments such as extreme temperature, drought, salt, and heavy metal stress at low concentration. Therefore, H2S homeostasis is very important in plant cells. The level of H2S in plant cells is regulated by its synthetic and degradative enzymes, L-/D-cysteine desulfhydrase (L-/D-DES), sulfite reductase (SiR), and cyanoalanine synthase (CAS), which are responsible for H2S synthesis, while cysteine synthase (CS) takes charge of the degradation of H2S, but its reverse reaction also can produce H2S. Here, after crude enzyme is extracted from plant tissues, the activities of L-/D-DES, SiR, CAS, and CS are measured by spectrophotometry, the aim is to further understand homeostasis of H2S in plant cells and its potential mechanisms.

The octahaem MccA is a haem c-copper sulfite reductase.

The six-electron reduction of sulfite to sulfide is the pivot point of the biogeochemical cycle of the element sulfur. The octahaem cytochrome c MccA (also known as SirA) catalyses this reaction for dissimilatory sulfite utilization by various bacteria. It is distinct from known sulfite reductases because it has a substantially higher catalytic activity and a relatively low reactivity towards nitrite. The mechanistic reasons for the increased efficiency of MccA remain to be elucidated. Here we show that anoxically purified MccA exhibited a 2- to 5.5-fold higher specific sulfite reductase activity than the enzyme isolated under oxic conditions. We determined the three-dimensional structure of MccA to 2.2 Å resolution by single-wavelength anomalous dispersion. We find a homotrimer with an unprecedented fold and haem arrangement, as well as a haem bound to a CX15CH motif. The heterobimetallic active-site haem 2 has a Cu(I) ion juxtaposed to a haem c at a Fe-Cu distance of 4.4 Å. While the combination of metals is reminiscent of respiratory haem-copper oxidases, the oxidation-labile Cu(I) centre of MccA did not seem to undergo a redox transition during catalysis. Intact MccA tightly bound SO2 at haem 2, a dehydration product of the substrate sulfite that was partially turned over due to photoreduction by X-ray irradiation, yielding the reaction intermediate SO. Our data show the biometal copper in a new context and function and provide a chemical rationale for the comparatively high catalytic activity of MccA.

The first echinoderm gamma-interferon-inducible lysosomal thiol reductase (GILT) identified from sea cucumber (Stichopus monotuberculatus).

Gamma-interferon-inducible lysosomal thiol reductase (GILT) has been described as a key enzyme that facilitating the processing and presentation of major histocompatibility complex class II-restricted antigen in mammals. In this study, the first echinoderm GILT named StmGILT was identified from sea cucumber (Stichopus monotuberculatus). The StmGILT cDNA is 1529 bp in length, containing a 5'-untranslated region (UTR) of 87 bp, a 3'-UTR of 674 bp and an open reading frame (ORF) of 768 bp that encoding a protein of 255 amino acids with a deduced molecular weight of 27.82 kDa and a predicted isoelectric point of 4.73. The putative StmGILT protein possesses all the main characteristics of known GILT proteins, including a signature sequence, a reductase active site CXXC, twelve conserved cysteines, and two potential N-linked glycosylation sites. For the gene structure, StmGILT contains four exons separated by three introns. In the promoter region of StmGILT gene, an NF-κB binding site and an IFN-γ activation site were found. The thiol reductase activity of recombinant StmGILT protein was also demonstrated in this study. In addition, the highest level of mRNA expression was noticed in coelomocytes of S. monotuberculatus. In in vitro experiments performed in coelomocytes, the expression of StmGILT mRNA was significantly up-regulated by lipopolysaccharides (LPS), inactivated bacteria or polyriboinosinic polyribocytidylic acid [poly (I:C)] challenge, suggested that the sea cucumber GILT might play critical roles in the innate immune defending against bacterial and viral infections.

Identification of gamma-interferon-inducible lysosomal thiol reductase (GILT) homologues in the fruit fly Drosophila melanogaster.

Gamma-interferon-inducible lysosomal thiol reductase (GILT) has been demonstrated to be involved in the immune response to bacterial challenge in various organisms. However, little is known about GILT function in innate immunity. Drosophila has been commonly used as a model for the study of the innate immune response of invertebrates. Here, we identify the CG9796, CG10157, and CG13822 genes of fruit fly Drosophila melanogaster as GILT homologues. All deduced Drosophila GILT coding sequences contained the major characteristic features of the GILT protein family: the GILT signature CQHGX2ECX2NX4C sequence and the active site CXXC or CXXS motif. The mRNA transcript levels of the Drosophila GILT genes were up-regulated after Gram-negative bacteria Escherichia coli DH5α infection. Moreover, a bacterial load assay showed that over-expression of Drosophila GILT in fat body or hemocytes led to a low bacterial colony number whereas knock-down of Drosophila GILT in fat body or hemocytes led to a high bacterial colony number when compared to a wild-type control. These results indicate that the Drosophila GILTs are very likely to play a role in the innate immune response upon bacterial challenge of Drosophila host defense. This study may provide the basis for further study on GILT function in innate immunity.

Disulfide bond generation in mammalian blood serum: detection and purification of quiescin-sulfhydryl oxidase.

A sensitive new plate-reader assay has been developed showing that adult mammalian blood serum contains circulating soluble sulfhydryl oxidase activity that can introduce disulfide bonds into reduced proteins with the reduction of oxygen to hydrogen peroxide. The activity was purified 5000-fold to >90% homogeneity from bovine serum and found by mass spectrometry to be consistent with the short isoform of quiescin-sulfhydryl oxidase 1 (QSOX1). This FAD-dependent enzyme is present at comparable activity levels in fetal and adult commercial bovine sera. Thus cell culture media that are routinely supplemented with either fetal or adult bovine sera will contain this facile catalyst of protein thiol oxidation. QSOX1 is present at approximately 25 nM in pooled normal adult human serum. Examination of the unusual kinetics of QSOX1 toward cysteine and glutathione at low micromolar concentrations suggests that circulating QSOX1 is unlikely to significantly contribute to the oxidation of these monothiols in plasma. However, the ability of QSOX1 to rapidly oxidize conformationally mobile protein thiols suggests a possible contribution to the redox status of exofacial and soluble proteins in blood plasma. Recent proteomic studies showing that plasma QSOX1 can be utilized in the diagnosis of pancreatic cancer and acute decompensated heart failure, together with the overexpression of this secreted enzyme in a number of solid tumors, suggest that the robust QSOX assay developed here may be useful in the quantitation of enzyme levels in a wide range of biological fluids.

Bacterial enzymes for dissimilatory sulfate reduction in a marine microbial mat (Black Sea) mediating anaerobic oxidation of methane.

Anaerobic oxidation of methane (AOM) with sulfate is catalysed by microbial consortia of archaea and bacteria affiliating with methanogens and sulfate-reducing Deltaproteobacteria respectively. There is evidence that methane oxidation is catalysed by enzymes related to those in methanogenesis, but the enzymes for sulfate reduction coupled to AOM have not been examined. We collected microbial mats with high AOM activity from a methane seep in the Black Sea. The mats consisted mainly of archaea of the ANME-2 group and bacteria of the Desulfosarcina-Desulfococcus group. Cell-free mat extract contained activities of enzymes involved in sulfate reduction to sulfide: ATP sulfurylase (adenylyl : sulfate transferase; Sat), APS reductase (Apr) and dissimilatory sulfite reductase (Dsr). We partially purified the enzymes by anion-exchange chromatography. The amounts obtained indicated that the enzymes are abundant in the mat, with Sat accounting for 2% of the soluble mat protein. N-terminal amino acid sequences of purified proteins suggested similarities to the corresponding enzymes of known species of sulfate-reducing bacteria. The deduced amino acid sequence of PCR-amplified genes of the Apr subunits is similar to that of Apr of the Desulfosarcina/Desulfococcus group. These results indicate that the major enzymes involved in sulfate reduction in the Back Sea microbial mats are of bacterial origin, most likely originating from the bacterial partner in the consortium.

Expression and crystallization of SeDsbA, SeDsbL and SeSrgA from Salmonella enterica serovar Typhimurium.

Pathogens require protein-folding enzymes to produce functional virulence determinants. These foldases include the Dsb family of proteins, which catalyze oxidative folding in bacteria. Bacterial disulfide catalytic processes have been well characterized in Escherichia coli K-12 and these mechanisms have been extrapolated to other organisms. However, recent research indicates that the K-12 complement of Dsb proteins is not common to all bacteria. Importantly, many pathogenic bacteria have an extended arsenal of Dsb catalysts that is linked to their virulence. To help to elucidate the process of oxidative folding in pathogens containing a wide repertoire of Dsb proteins, Salmonella enterica serovar Typhimurium has been focused on. This Gram-negative bacterium contains three DsbA proteins: SeDsbA, SeDsbL and SeSrgA. Here, the expression, purification, crystallization and preliminary diffraction analysis of these three proteins are reported. SeDsbA, SeDsbL and SeSrgA crystals diffracted to resolution limits of 1.55, 1.57 and 2.6 A and belonged to space groups P2(1), P2(1)2(1)2 and C2, respectively.

Purification, crystallization and preliminary X-ray analysis of adenylylsulfate reductase from Desulfovibrio vulgaris Miyazaki F.

Sulfur in its various oxidation states is used for energy conservation in many microorganisms. Adenylylsulfate reductase is a key enzyme in the sulfur-reduction pathway of sulfate-reducing bacteria. The adenylylsulfate reductase from Desulfovibrio vulgaris Miyazaki F has been purified and crystallized at 277 K using the vapour-diffusion method with ammonium sulfate as the precipitating agent. A data set was collected to 1.7 A resolution from a single crystal at 100 K using synchrotron radiation. The crystal belonged to space group P3(1), with unit-cell parameters a = b = 125.93, c = 164.24 A. The crystal contained two molecules per asymmetric unit, with a Matthews coefficient (V(M)) of 4.02 A(3) Da(-1); the solvent content was estimated to be 69.4%.

Identification of two inactive forms of the central sulfur cycle protein SoxYZ of Paracoccus pantotrophus.

The central protein of the sulfur-oxidizing enzyme system of Paracoccus pantotrophus, SoxYZ, reacts with three different Sox proteins. Its active site Cys110(Y) is on the carboxy-terminus of the SoxY subunit. SoxYZ "as isolated" consisted mainly of the catalytically inactive SoxY-Y(Z)(2) heterotetramer linked by a Cys110(Y)-Cys110(Y) interprotein disulfide. Sulfide activated SoxYZ "as isolated" 456-fold, reduced the disulfide, and yielded an active SoxYZ heterodimer. The reductant tris(2-carboxyethyl)phosphine (TCEP) inactivated SoxYZ. This form was not re-activated by sulfide, which identified it as a different inactive form. In analytical gel filtration, the elution of "TCEP-treated" SoxYZ was retarded compared to active SoxYZ, indicating a conformational change. The possible enzymes involved in the re-activation of each inactive form of SoxYZ are discussed.

The thioredoxin homolog YbbN functions as a chaperone rather than as an oxidoreductase.

Escherichia coli contains two thioredoxins, Trx1 and Trx2, and a thioredoxin-like protein, YbbN, which presents a strong homology in its N-terminal part with thioredoxins, and possesses a 20kDa C-terminal part of unknown function. We reported previously that YbbN displays both protein oxido-reductase and chaperone properties in vitro. In this study, we show that an ybbN-deficient strain displays an increased sensitivity to thermal stress but not to oxidative stress, a normal redox state of its cellular proteins but a decreased expression of several cytoplasmic proteins, including EF-Tu, DnaK, GroEL, trigger factor and several Krebs cycle enzymes, suggesting that the chaperone properties of YbbN are more important in vivo than its redox properties. YbbN specifically interacts with DnaK and GroEL, as shown by reverse purification. It increases 4-fold the rate of protein renaturation in vitro by the DnaK chaperone machine, suggesting that it cooperates with DnaK for the optimal expression of several cytoplasmic proteins.

Bacterial sulfite dehydrogenases in organotrophic metabolism: separation and identification in Cupriavidus necator H16 and in Delftia acidovorans SPH-1.

The utilization of organosulfonates as carbon sources by aerobic or nitrate-reducing bacteria usually involves a measurable, uncharacterized sulfite dehydrogenase. This is tacitly assumed to be sulfite : ferricytochrome-c oxidoreductase [EC 1.8.2.1], despite negligible interaction with (eukaryotic) cytochrome c: the enzyme is assayed at high specific activity with ferricyanide as electron acceptor. Purified periplasmic sulfite dehydrogenases (SorAB, SoxCD) are known from chemoautotrophic growth and are termed 'sulfite oxidases' by bioinformatic services. The catalytic unit (SorA, SoxC; termed 'sulfite oxidases' cd02114 and cd02113, respectively) binds a molybdenum-cofactor (Moco), and involves a cytochrome c (SorB, SoxD) as electron acceptor. The genomes of several bacteria that express a sulfite dehydrogenase during heterotrophic growth contain neither sorAB nor soxCD genes; others contain at least four paralogues, for example Cupriavidus necator H16, which is known to express an inducible sulfite dehydrogenase during growth with taurine (2-aminoethanesulfonate). This soluble enzyme was enriched 320-fold in four steps. The 40 kDa protein (denatured) had an N-terminal amino acid sequence which started at position 42 of the deduced sequence of H16_B0860 (termed 'sulfite oxidase' cd02114), which we named SorA. The neighbouring gene is an orthologue of sorB, and the sorAB genes were co-transcribed. Cell fractionation showed SorA to be periplasmic. The corresponding enzyme in Delftia acidovorans SPH-1 was enriched 270-fold, identified as Daci_0055 (termed 'sulfite oxidase' cd02110) and has a cytochrome c encoded downstream. We presume, from genomic data for bacteria and archaea, that there are several subgroups of sulfite dehydrogenases, which all contain a Moco, and transfer electrons to a specific cytochrome c.

The role of 5'-adenylylsulfate reductase in the sulfur assimilation pathway of soybean: molecular cloning, kinetic characterization, and gene expression.

Soybean seeds are a major source of protein, but contain low levels of sulfur-containing amino acids. With the objective of studying the sulfur assimilation pathway of soybean, a full-length cDNA clone for 5'-adenylylsulfate reductase (APS reductase) was isolated and characterized. The cDNA clone contained an open reading frame of 1414 bp encoding a 52 kDa protein with a N-terminal chloroplast/plastid transit peptide. Southern blot analysis of genomic DNA indicated that the APS reductase in soybean is encoded by a small multigene family. Biochemical characterization of the heterologously expressed and purified protein shows that the clone encoded a functional APS reductase. Although expressed in tissues throughout the plant, these analyses established an abundant expression of the gene and activity of the encoded protein in the early developmental stages of soybean seed, which declined with seed maturity. Sulfur and phosphorus deprivation increased this expression level, while nitrogen starvation repressed APS reductase mRNA transcript and protein levels. Cold-treatment increased expression and the total activity of APS reductase in root tissues. This study provides insight into the sulfur assimilation pathway of this nutritionally important legume.

Ultrasonic assisted protein enzymatic digestion for fast protein identification by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Sonoreactor versus ultrasonic probe.

Two different ultrasonic energy sources, the sonoreactor and the ultrasonic probe, are compared for enzymatic digestion of proteins for protein identification by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) using the peptide mass fingerprint (PMF) procedure. Variables such as (i) trypsin/protein ratio; (ii) sonication time; (iii) ultrasound amplitude; and (iv) protein concentration are studied and compared. As a general rule, the trypsin/protein ratio and the minimum protein concentration successfully digested are similar with both ultrasonic energy sources. Results showed that the time needed to digest proteins was shorter with the ultrasonic probe, 60s versus 120s, for the same amplitude of sonication, 50%. However, lower standard deviations and cleaner MALDI-TOF-MS spectra were obtained with the sonoreactor. In addition, the sonoreactor device provided higher sample throughput (6 samples for the sonoreactor versus 1 sample for the ultrasonic probe) and easier sample handling for lower sample volumes (25 microl). Finally, a comparison of both methodologies for the specific identification of the adenylylsulphate reductase alfa subunit from a complex protein mixture from Desulfovibrio desulfuricans ATCC 27774 was done as a proof of the procedure.

Insights into the metabolism of elemental sulfur by the hyperthermophilic archaeon Pyrococcus furiosus: characterization of a coenzyme A- dependent NAD(P)H sulfur oxidoreductase.

The hyperthermophilic archaeon Pyrococcus furiosus uses carbohydrates as a carbon source and produces acetate, CO2, and H2 as end products. When S(0) is added to a growing culture, within 10 min the rate of H2 production rapidly decreases and H(2)S is detected. After 1 hour cells contain high NADPH- and coenzyme A-dependent S(0) reduction activity (0.7 units/mg, 85 degrees C) located in the cytoplasm. The enzyme responsible for this activity was purified to electrophoretic homogeneity (specific activity, 100 units/mg) and is termed NAD(P)H elemental sulfur oxidoreductase (NSR). NSR is a homodimeric flavoprotein (M(r), 100,000) and is encoded by PF1186. This designation was previously assigned to the gene encoding an enzyme that reduces coenzyme A disulfide, which is a side reaction of NSR. Whole-genome DNA microarray and quantitative PCR analyses showed that the expression of NSR is up-regulated up to sevenfold within 10 min of S(0) addition. This primary response to S(0) also involves the up-regulation (>16-fold) of a 13-gene cluster encoding a membrane-bound oxidoreductase (MBX). The cluster encoding MBX is proposed to replace the homologous 14-gene cluster that encodes the ferredoxin-oxidizing, H2-evolving membrane-bound hydrogenase (MBH), which is down-regulated >12-fold within 10 min of S(0) addition. Although an activity for MBX could not be demonstrated, it is proposed to conserve energy by oxidizing ferredoxin and reducing NADP, which is used by NSR to reduce S(0). A secondary response to S(0) is observed 30 min after S(0) addition and includes the up-regulation of genes encoding proteins involved in amino acid biosynthesis and iron metabolism, as well as two so-called sulfur-induced proteins termed SipA and SipB. This novel S(0)-reducing system involving NSR and MBX has been found so far only in the heterotrophic Thermococcales and is in contrast to the cytochrome- and quinone-based S(0)-reducing system in autotrophic archaea and bacteria.

New findings for in-gel digestion accelerated by high-intensity focused ultrasound for protein identification by matrix-assisted laser desorption ionization time-of-flight mass spectrometry.

New findings in sample treatment based on high-intensity focused ultrasound (HIFU) for protein digestion after polyacrylamide gel electrophoresis separation are presented. The following variables were studied: (i) sample volume; (ii) sonotrode diameter; (iii) previous protein denaturation; (iv) cooling; (v) enzyme concentration; and (vi) protein concentration. Results showed that positive protein identification could be done after protein separation by gel electrophoresis through peptide mass fingerprint (PMF) in a volume as low as 25 microL. The time needed was less than 2 min and no cooling was necessary. The importance of the sonotrode diameter was negligible. On the other hand, protein denaturation before sonication was a trade-off for the success of procedure here described. The protein coverage was raised from 5 to 30%, and the number of peptides matching the proteins was also increased in a percentage ranging 10-100% when the classical overnight treatment is compared with the proposed HIFU procedure. The minimum amount of protein that can be identified using the HIFU sample treatment by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) was 0.06 microg. The lower concentration of trypsin successfully used to obtain an adequate protein digestion was 3.6 microg/mL.

Siro(haem)amide in Allochromatium vinosum and relevance of DsrL and DsrN, a homolog of cobyrinic acid a,c-diamide synthase, for sulphur oxidation.

In the purple sulphur bacterium Allochromatium vinosum, the prosthetic group of dissimilatory sulphite reductase (DsrAB) was identified as siroamide, an amidated form of the classical sirohaem. The genes dsrAB are the first two of a large cluster of genes necessary for the oxidation of sulphur globules stored intracellularly during growth on sulphide and thiosulphate. DsrN is homologous to cobyrinic acid a,c diamide synthase and may therefore catalyze glutamine-dependent amidation of sirohaem. Indeed, an A. vinosumDeltadsrN in frame deletion mutant showed a significantly reduced sulphur oxidation rate that was fully restored upon complementation with dsrN in trans. Sulphite reductase was still present in the DeltadsrN mutant. DsrL is a homolog of the small subunits of bacterial glutamate synthases and was proposed to deliver glutamine for sirohaem amidation. However, recombinant DsrL does not exhibit glutamate synthase activity nor does the gene complement a glutamate synthase-deficient Escherichia coli strain. Deletion of dsrL showed that the encoded protein is absolutely essential for sulphur oxidation in A. vinosum.

Crystal structure and desulfurization mechanism of 2'-hydroxybiphenyl-2-sulfinic acid desulfinase.

The desulfurization of dibenzothiophene in Rhodococcus erythropolis is catalyzed by two monooxygenases, DszA and DszC, and a desulfinase, DszB. In the last step of this pathway, DszB hydrolyzes 2'-hydroxybiphenyl-2-sulfinic acid into 2-hydroxybiphenyl and sulfite. We report on the crystal structures of DszB and an inactive mutant of DszB in complex with substrates at resolutions of 1.8A or better. The overall fold of DszB is similar to those of periplasmic substrate-binding proteins. In the substrate complexes, biphenyl rings of substrates are recognized by extensive hydrophobic interactions with the active site residues. Binding of substrates accompanies structural changes of the active site loops and recruits His(60) to the active site. The sulfinate group of bound substrates forms hydrogen bonds with side chains of Ser(27), His(60), and Arg(70), each of which is shown by site-directed mutagenesis to be essential for the activity. In our proposed reaction mechanism, Cys(27) functions as a nucleophile and seems to be activated by the sulfinate group of substrates, whereas His(60) and Arg(70) orient the syn orbital of sulfinate oxygen to the sulfhydryl hydrogen of Cys(27) and stabilize the negatively charged reaction intermediate. Cys, His, and Arg residues are conserved in putative proteins homologous to DszB, which are presumed to constitute a new family of desulfinases.

Structural insights into sulfite oxidase deficiency.

Sulfite oxidase deficiency is a lethal genetic disease that results from defects either in the genes encoding proteins involved in molybdenum cofactor biosynthesis or in the sulfite oxidase gene itself. Several point mutations in the sulfite oxidase gene have been identified from patients suffering from this disease worldwide. Although detailed biochemical analyses have been carried out on these mutations, no structural data could be obtained because of problems in crystallizing recombinant human and rat sulfite oxidases and the failure to clone the chicken sulfite oxidase gene. We synthesized the gene for chicken sulfite oxidase de novo, working backward from the amino acid sequence of the native chicken liver enzyme by PCR amplification of a series of 72 overlapping primers. The recombinant protein displayed the characteristic absorption spectrum of sulfite oxidase and exhibited steady state and rapid kinetic parameters comparable with those of the tissue-derived enzyme. We solved the crystal structures of the wild type and the sulfite oxidase deficiency-causing R138Q (R160Q in humans) variant of recombinant chicken sulfite oxidase in the resting and sulfate-bound forms. Significant alterations in the substrate-binding pocket were detected in the structure of the mutant, and a comparison between the wild type and mutant protein revealed that the active site residue Arg-450 adopts different conformations in the presence and absence of bound sulfate. The size of the binding pocket is thereby considerably reduced, and its position relative to the cofactor is shifted, causing an increase in the distance of the sulfur atom of the bound sulfate to the molybdenum.

Novel genes of the dsr gene cluster and evidence for close interaction of Dsr proteins during sulfur oxidation in the phototrophic sulfur bacterium Allochromatium vinosum.

Seven new genes designated dsrLJOPNSR were identified immediately downstream of dsrABEFHCMK, completing the dsr gene cluster of the phototrophic sulfur bacterium Allochromatium vinosum D (DSM 180(T)). Interposon mutagenesis proved an essential role of the encoded proteins for the oxidation of intracellular sulfur, an obligate intermediate during the oxidation of sulfide and thiosulfate. While dsrR and dsrS encode cytoplasmic proteins of unknown function, the other genes encode a predicted NADPH:acceptor oxidoreductase (DsrL), a triheme c-type cytochrome (DsrJ), a periplasmic iron-sulfur protein (DsrO), and an integral membrane protein (DsrP). DsrN resembles cobyrinic acid a,c-diamide synthases and is probably involved in the biosynthesis of siro(heme)amide, the prosthetic group of the dsrAB-encoded sulfite reductase. The presence of most predicted Dsr proteins in A. vinosum was verified by Western blot analysis. With the exception of the constitutively present DsrC, the formation of Dsr gene products was greatly enhanced by sulfide. DsrEFH were purified from the soluble fraction and constitute a soluble alpha(2)beta(2)gamma(2)-structured 75-kDa holoprotein. DsrKJO were purified from membranes pointing at the presence of a transmembrane electron-transporting complex consisting of DsrKMJOP. In accordance with the suggestion that related complexes from dissimilatory sulfate reducers transfer electrons to sulfite reductase, the A. vinosum Dsr complex is copurified with sulfite reductase, DsrEFH, and DsrC. We therefore now have an ideal and unique possibility to study the interaction of sulfite reductase with other proteins and to clarify the long-standing problem of electron transport from and to sulfite reductase, not only in phototrophic bacteria but also in sulfate-reducing prokaryotes.