Discovery of the Lanthipeptide Curvocidin†and Structural Insights into its Trifunctional Synthetase CuvL.

Lanthipeptides are ribosomally-synthesized natural products from bacteria featuring stable thioether-crosslinks and various bioactivities. Herein, we report on a new clade of tricyclic class-IV lanthipeptides with curvocidin from Thermomonospora curvata as its first representative. We obtained crystal structures of the corresponding lanthipeptide synthetase CuvL that showed a circular arrangement of its kinase, lyase and cyclase domains, forming a central reaction chamber for the iterative substrate processing involving nine catalytic steps. The combination of experimental data and artificial intelligence-based structural models identified the N-terminal subdomain of the kinase domain as the primary site of substrate recruitment. The ribosomal precursor peptide of curvocidin employs an amphipathic α-helix in its leader region as an anchor to CuvL, while its substrate core shuttles within the central reaction chamber. Our study thus reveals general principles of domain organization and substrate recruitment of class-IV and class-III lanthipeptide synthetases.

Double-Cubane [8Fe9S] Clusters: A Novel Nitrogenase-Related Cofactor in Biology.

Three different types of electron-transferring metallo-ATPases are able to couple ATP hydrolysis to the reduction of low-potential metal sites, thereby energizing an electron. Besides the Fe-protein known from nitrogenase and homologous enzymes, two other kinds of ATPase with different scaffolds and cofactors are used to achieve a unidirectional, energetic, uphill electron transfer to either reduce inactive Co-corrinoid-containing proteins (RACE-type activators) or a second iron-sulfur cluster-containing enzyme of a unique radical enzymes family (archerases). We have found a new cofactor in the latter enzyme family, that is, a double-cubane cluster with two [4Fe4S] subclusters bridged by a sulfido ligand. An enzyme containing this cofactor catalyzes the ATP-dependent reduction of small molecules, including acetylene. Thus, enzymes containing the double-cubane cofactor are analogous in function and share some structural features with nitrogenases.

Site-Resolved Observation of Vibrational Energy Transfer Using a Genetically Encoded Ultrafast Heater.

Allosteric information transfer in proteins has been linked to distinct vibrational energy transfer (VET) pathways in a number of theoretical studies. Experimental evidence for such pathways, however, is sparse because site-selective injection of vibrational energy into a protein, that is, localized heating, is required for their investigation. Here, we solved this problem by the site-specific incorporation of the non-canonical amino acid ß-(1-azulenyl)-l-alanine (AzAla) through genetic code expansion. As an exception to Kasha's rule, AzAla undergoes ultrafast internal conversion and heating after S1 excitation while upon S2 excitation, it serves as a fluorescent label. We equipped PDZ3, a protein interaction domain of postsynaptic density protein 95, with this ultrafast heater at two distinct positions. We indeed observed VET from the incorporation sites in the protein to a bound peptide ligand on the picosecond timescale by ultrafast IR spectroscopy. This approach based on genetically encoded AzAla paves the way for detailed studies of VET and its role in a wide range of proteins.

Photoactivatable Mussel-Based Underwater Adhesive Proteins by an Expanded Genetic Code.

Marine mussels exhibit potent underwater adhesion abilities under hostile conditions by employing 3,4-dihydroxyphenylalanine (DOPA)-rich mussel adhesive proteins (MAPs). However, their recombinant production is a major biotechnological challenge. Herein, a novel strategy based on genetic code expansion has been developed by engineering efficient aminoacyl-transfer RNA synthetases (aaRSs) for the photocaged noncanonical amino acid ortho-nitrobenzyl DOPA (ONB-DOPA). The engineered ONB-DOPARS enables in vivo production of MAP type 5 site-specifically equipped with multiple instances of ONB-DOPA to yield photocaged, spatiotemporally controlled underwater adhesives. Upon exposure to UV light, these proteins feature elevated wet adhesion properties. This concept offers new perspectives for the production of recombinant bioadhesives.

Substrate-induced radical formation in 4-hydroxybutyryl coenzyme A dehydratase from Clostridium aminobutyricum.

4-Hydroxybutyryl-coenzyme A (CoA) dehydratase (4HBD) from Clostridium aminobutyricum catalyzes the reversible dehydration of 4-hydroxybutyryl-CoA to crotonyl-CoA and the irreversible isomerization of vinylacetyl-CoA to crotonyl-CoA. 4HBD is an oxygen-sensitive homotetrameric enzyme with one [4Fe-4S](2+) cluster and one flavin adenine dinucleotide (FAD) in each subunit. Upon the addition of crotonyl-CoA or the analogues butyryl-CoA, acetyl-CoA, and CoA, UV-visible light and electron paramagnetic resonance (EPR) spectroscopy revealed an internal one-electron transfer to FAD and the [4Fe-4S](2+) cluster prior to hydration. We describe an active recombinant 4HBD and variants produced in Escherichia coli. The variants of the cluster ligands (H292C [histidine at position 292 is replaced by cysteine], H292E, C99A, C103A, and C299A) had no measurable dehydratase activity and were composed of monomers, dimers, and tetramers. Variants of other potential catalytic residues were composed only of tetramers and exhibited either no measurable (E257Q, E455Q, and Y296W) hydratase activity or <1% (Y296F and T190V) dehydratase activity. The E455Q variant but not the Y296F or E257Q variant displayed the same spectral changes as the wild-type enzyme after the addition of crotonyl-CoA but at a much lower rate. The results suggest that upon the addition of a substrate, Y296 is deprotonated by E455 and reduces FAD to FADH·, aided by protonation from E257 via T190. In contrast to FADH·, the tyrosyl radical could not be detected by EPR spectroscopy. FADH· appears to initiate the radical dehydration via an allylic ketyl radical that was proposed 19 years ago. The mode of radical generation in 4HBD is without precedent in anaerobic radical chemistry. It differs largely from that in enzymes, which use coenzyme B12, S-adenosylmethionine, ATP-driven electron transfer, or flavin-based electron bifurcation for this purpose.

The ferredoxin-like domain of the activating enzyme is required for generating a lasting glycyl radical in 4-hydroxyphenylacetate decarboxylase.

4-Hydroxyphenylacetate decarboxylase-activating enzyme (4Hpad-AE) uses S-adenosylmethionine (SAM or AdoMet) and a [4Fe-4S] ²âº/⁺cluster (RS cluster) to generate a stable glycyl radical on the decarboxylase. 4Hpad-AE might bind up to two auxiliary [4Fe-4S] clusters coordinated by a ferredoxin-like insert C-terminal to the RS cluster-binding motif. Except for the AEs of pyruvate formate-lyase and anaerobic ribonucleotide reductase, all glycyl radical-activating enzymes possess a similar ferredoxin-like domain, whose functional role is still poorly understood. To assess the role of the putative ferredoxin clusters from 4Hpad-AE, we combined biochemical and spectroscopic methods to characterize a truncated version of the protein (Δ66-AE) devoid of the ferredoxin-like domain. We found that Δ66-AE is stable, harbors a fully active RS cluster and can activate the decarboxylase. From the similar cleavage rates for S-adenosylmethionine of Δ66-AE and wild-type AE, we infer the reactivity of the RS cluster is unperturbed by the absence of the ferredoxin-like domain. Thus, the auxiliary clusters are not required as electron conduit to the RS cluster for effective reductive cleavage of SAM. The activation of the decarboxylase by Δ66-AE is almost as fast as with wild-type AE, but the generated glycyl radical is short living. We postulate that the ferredoxin-like domain is not required for SAM-dependent glycyl radical generation in the decarboxylase, but is necessary for producing a lasting glycyl radical.

Enzyme catalyzed radical dehydrations of hydroxy acids.

BACKGROUND: The steadily increasing field of radical biochemistry is dominated by the large family of S-adenosylmethionine dependent enzymes, the so-called radical SAM enzymes, of which several new members are discovered every year. Here we report on 2- and 4-hydroxyacyl-CoA dehydratases which apply a very different method of radical generation. In these enzymes ketyl radicals are formed by one-electron reduction or oxidation and are recycled after each turnover without further energy input. Earlier reviews on 2-hydroxyacyl-CoA dehydratases were published in 2004 [J. Kim, M. Hetzel, C.D. Boiangiu, W. Buckel, FEMS Microbiol. Rev. 28 (2004) 455-468. W. Buckel, M. Hetzel, J. Kim, Curr. Opin. Chem. Biol. 8 (2004) 462-467.] SCOPE OF REVIEW: The review focuses on four types of 2-hydroxyacyl-CoA dehydratases that are involved in the fermentation of amino acids by anaerobic bacteria, especially clostridia. These enzymes require activation by one-electron transfer from an iron-sulfur protein driven by hydrolysis of ATP. The review further describes the proposed mechanism that is highlighted by the identification of the allylic ketyl radical intermediate and the elucidation of the crystal structure of 2-hydroxyisocapryloyl-CoA dehydratase. With 4-hydroxybutyryl-CoA dehydratase the crystal structure, the complete stereochemistry and the function of several conserved residues around the active site could be identified. Finally potential biotechnological applications of the radical dehydratases are presented. GENERAL SIGNIFICANCE: The action of the activator as an 'Archerase' shooting electrons into difficultly reducible acceptors becomes an emerging principle in anaerobic metabolism. The dehydratases may provide useful tools in biotechnology. This article is part of a Special Issue entitled: Radical SAM enzymes and Radical Enzymology.

Catalytic mechanism of the glycyl radical enzyme 4-hydroxyphenylacetate decarboxylase from continuum electrostatic and QC/MM calculations.

Using continuum electrostatics and QC/MM calculations, we investigate the catalytic cycle of the glycyl radical enzyme 4-hydroxyphenylacetate decarboxylase, an enzyme involved in the fermentative production of p-cresol from tyrosine in clostridia. On the basis of our calculations, we propose a five-step mechanism for the reaction. In the first step, the substrate 4-hydroxyphenylacetate is activated by an unusual concerted abstraction of an electron and a proton. Namely, Cys503 radical abstracts an electron from the substrate and Glu637 abstracts a proton. Thus in total, a hydrogen atom is abstracted from the substrate. In the second step, the carboxylic group readily splits off from the phenoxy-acetate radical anion to give carbon dioxide. This decarboxylation step is coupled to a proton transfer from Glu637 back to the phenolic hydroxyl group which leads to a p-hydroxybenzyl radical. The remaining steps of the reaction involve a rotation of the Cys503 side chain followed by a proton transfer from Glu505 to Cys503 and a hydrogen atom transfer from Cys503 to the p-hydroxybenzyl radical to form p-cresol. The calculated mechanism agrees with experimental data suggesting that both Cys503 and Glu637 are essential for the catalytic function of 4-hydroxyphenylacetate decarboxylase and that the substrate requires a hydroxyl group in para-position to the acetate moiety.

Determinants of substrate specificity and biochemical properties of the sn-glycerol-3-phosphate ATP binding cassette transporter (UgpB-AEC2 ) of Escherichia coli.

Under phosphate starvation conditions, Escherichia coli can utilize sn-glycerol-3-phosphate (G3P) and G3P diesters as phosphate source when transported by an ATP binding cassette importer composed of the periplasmic binding protein, UgpB, the transmembrane subunits, UgpA and UgpE, and a homodimer of the nucleotide binding subunit, UgpC. The current knowledge on the Ugp transporter is solely based on genetic evidence and transport assays using intact cells. Thus, we set out to characterize its properties at the level of purified protein components. UgpB was demonstrated to bind G3P and glycerophosphocholine with dissociation constants of 0.68 ± 0.02 µM and 5.1 ± 0.3 µM, respectively, while glycerol-2-phosphate (G2P) is not a substrate. The crystal structure of UgpB in complex with G3P was solved at 1.8 Å resolution and revealed the interaction with two tryptophan residues as key to the preferential binding of linear G3P in contrast to the branched G2P. Mutational analysis validated the crucial role of Trp-169 for G3P binding. The purified UgpAEC2 complex displayed UgpB/G3P-stimulated ATPase activity in proteoliposomes that was neither inhibited by phosphate nor by the signal transducing protein PhoU or the phosphodiesterase UgpQ. Furthermore, a hybrid transporter composed of MalFG-UgpC could be functionally reconstituted while a UgpAE-MalK complex was unstable.

Broadened T-cell repertoire diversity in ivIg-treated SLE patients is also related to the individual status of regulatory T-cells.

PURPOSE: Intravenous IgG (ivIg) is a therapeutic alternative for lupus erythematosus, the mechanism of which remains to be fully understood. Here we investigated whether ivIg affects two established sub-phenotypes of SLE, namely relative oligoclonality of circulating T-cells and reduced activity of CD4 + Foxp3+ regulatory T-cells (Tregs) reflected by lower CD25 surface density. METHODS: We conducted a longitudinal study of 15 lupus patients (14 with SLE and one with discoid LE) treated with ivIg in cycles of 2-6 consecutive monthly infusions. Among these 15 patients, 10 responded to ivIg therapy with clear clinical improvement. We characterized Tregs and determined TCR spectratypes of four Vß families with reported oligoclonality. Cell counts, cytometry and TCR spectratypes were obtained from peripheral blood at various time points before, during and after ivIg treatment. T-cell oligoclonality was assessed as Vß-familywise repertoire perturbation, calculated for each patient in respect to an individual reference profile averaged over all available time points. RESULTS: For 11 out of 15 patients, average Vß1/Vß2/Vß11/Vß14 repertoires were less perturbed under than outside ivIg therapy. The four exceptions with relatively increased average perturbation during ivIg therapy included three patients who failed to respond clinically to an ivIg therapy cycle. Patients' Treg CD25 surface density (cytometric MFI) was clearly reduced when compared to healthy controls, but not obviously influenced by ivIg. However, patients' average Treg CD25 MFI was found negatively correlated with both Vß11 and Vß14 perturbations measured under ivIg therapy. CONCLUSIONS: This indicates a role of active Tregs in the therapeutic effect of ivIg.

4-Hydroxyphenylacetate decarboxylase activating enzyme catalyses a classical S-adenosylmethionine reductive cleavage reaction.

4-Hydroxyphenylacetate decarboxylase (4Hpad) is an Fe/S cluster containing glycyl radical enzyme (GRE), which catalyses the last step of tyrosine fermentation in clostridia, generating the bacteriostatic p-cresol. The respective activating enzyme (4Hpad-AE) displays two cysteine-rich motifs in addition to the classical S-adenosylmethionine (SAM) binding cluster (RS cluster) motif. These additional motifs are also present in other glycyl radical activating enzymes (GR-AE) and it has been postulated that these orthologues may use an alternative SAM homolytic cleavage mechanism, generating a putative 3-amino-3-carboxypropyl radical and 5'-deoxy-5'-(methylthio)adenosine but not a 5'-deoxyadenosyl radical and methionine. 4Hpad-AE produced from a codon-optimized synthetic gene binds a maximum of two [4Fe-4S](2+/+) clusters as revealed by EPR and Mössbauer spectroscopy. The enzyme only catalyses the turnover of SAM under reducing conditions, and the reaction products were identified as 5'-deoxyadenosine (quenched form of 5'-deoxyadenosyl radical) and methionine. We demonstrate that the 5'-deoxyadenosyl radical is the activating agent for 4Hpad through p-cresol formation and correlation between the production of 5'-deoxyadenosine and the generation of glycyl radical in 4Hpad. Therefore, we conclude that 4Hpad-AE catalyses a classical SAM-dependent glycyl radical formation as reported for GR-AE without auxiliary clusters. Our observation casts doubt on the suggestion that GR-AE containing auxiliary clusters catalyse the alternative cleavage reaction detected for glycerol dehydratase activating enzyme.

Structural basis for a Kolbe-type decarboxylation catalyzed by a glycyl radical enzyme.

4-Hydroxyphenylacetate decarboxylase is a [4Fe-4S] cluster containing glycyl radical enzyme proposed to use a glycyl/thiyl radical dyad to catalyze the last step of tyrosine fermentation in clostridia. The decarboxylation product p-cresol (4-methylphenol) is a virulence factor of the human pathogen Clostridium difficile . Here we describe the crystal structures at 1.75 and 1.81 Å resolution of substrate-free and substrate-bound 4-hydroxyphenylacetate decarboxylase from the related Clostridium scatologenes . The structures show a (ßγ)(4) tetramer of heterodimers composed of a catalytic ß-subunit harboring the putative glycyl/thiyl dyad and a distinct small γ-subunit with two [4Fe-4S] clusters at 40 Å distance from the active site. The γ-subunit comprises two domains displaying pseudo-2-fold symmetry that are structurally related to the [4Fe-4S] cluster-binding scaffold of high-potential iron-sulfur proteins. The N-terminal domain coordinates one cluster with one histidine and three cysteines, and the C-terminal domain coordinates the second cluster with four cysteines. Whereas the C-terminal cluster is buried in the ßγ heterodimer interface, the N-terminal cluster is not part of the interface. The previously postulated decarboxylation mechanism required the substrate's hydroxyl group in the vicinity of the active cysteine residue. In contrast to expectation, the substrate-bound state shows a direct interaction between the substrate's carboxyl group and the active site Cys503, while His536 and Glu637 at the opposite side of the active site pocket anchor the hydroxyl group. This state captures a possible catalytically competent complex and suggests a Kolbe-type decarboxylation for p-cresol formation.

Crystal structure of the human transcription elongation factor DSIF hSpt4 subunit in complex with the hSpt5 dimerization interface.

The eukaryotic transcription elongation factor DSIF [DRB (5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole) sensitivity-inducing factor] is composed of two subunits, hSpt4 and hSpt5, which are homologous to the yeast factors Spt4 and Spt5. DSIF is involved in regulating the processivity of RNA polymerase II and plays an essential role in transcriptional activation of eukaryotes. At several eukaryotic promoters, DSIF, together with NELF (negative elongation factor), leads to promoter-proximal pausing of RNA polymerase II. In the present paper we describe the crystal structure of hSpt4 in complex with the dimerization region of hSpt5 (amino acids 176-273) at a resolution of 1.55 A (1 A=0.1 nm). The heterodimer shows high structural similarity to its homologue from Saccharomyces cerevisiae. Furthermore, hSpt5-NGN is structurally similar to the NTD (N-terminal domain) of the bacterial transcription factor NusG. A homologue for hSpt4 has not yet been found in bacteria. However, the archaeal transcription factor RpoE" appears to be distantly related. Although a comparison of the NusG-NTD of Escherichia coli with hSpt5 revealed a similarity of the three-dimensional structures, interaction of E. coli NusG-NTD with hSpt4 could not be observed by NMR titration experiments. A conserved glutamate residue, which was shown to be crucial for dimerization in yeast, is also involved in the human heterodimer, but is substituted for a glutamine residue in Escherichia coli NusG. However, exchanging the glutamine for glutamate proved not to be sufficient to induce hSpt4 binding.

Nickel, Iron, Sulfur Sites.

Enzymes relying on the interplay of nickel, iron, and sulfur in their active sites are used by prokaryotes to catalyze reactions driving the global carbon and hydrogen cycles. The three enzymes, [NiFe] hydrogenases, Ni,Fe-containing carbon monoxide dehydrogenases and acetyl-CoA synthases share an ancient origin possibly derived from abiotic processes. Although their active sites have different compositions and assemble Ni, Fe, and S in different ways and for different purposes, they share a central role of Ni in substrate binding and activation, with sulfur linking the Ni ion to one or more Fe ions, which, although indispensable for function, supports the catalytic process in less understood ways. The review gives a short overview on the properties of the three individual enzymes highlighting their parallels and differences.

Low frequency of CD4+CD25+ Treg in SLE patients: a heritable trait associated with CTLA4 and TGFbeta gene variants.

BACKGROUND: CD4+CD25+ regulatory T cells play an essential role in maintaining immune homeostasis and preventing autoimmunity. Therefore, defects in Treg development, maintenance or function have been associated with several human autoimmune diseases including Systemic Lupus Erythematosus (SLE), a systemic autoimmune disease characterized by loss of tolerance to nuclear components and significantly more frequent in females. RESULTS: To investigate the involvement of Treg in SLE pathogenesis, we determined the frequency of CD4+CD25+CD45RO+ T cells, which encompass the majority of Treg activity, in the PBMC of 148 SLE patients (76 patients were part of 54 families), 166 relatives and 117 controls. SLE patients and their relatives were recruited in several Portuguese hospitals and through the Portuguese Lupus Association. Control individuals were blood donors recruited from several regional blood donor centers. Treg frequency was significantly lower in SLE patients than healthy controls (z = -6.161, P < 0.00001) and intermediate in the relatives' group. Remarkably, this T cell subset was also lower in females, most strikingly in the control population (z = 4.121, P < 0.001). We further ascertained that the decreased frequency of Treg in SLE patients resulted from the specific reduction of bona fide FOXP3+CD4+CD25+ Treg. Treg frequency was negatively correlated with SLE activity index (SLEDAI) and titers of serum anti-dsDNA antibodies. Both Treg frequency and disease activity were modulated by IVIg treatment in a documented SLE case. The segregation of Treg frequency within the SLE families was indicative of a genetic trait. Candidate gene analysis revealed that specific variants of CTLA4 and TGFbeta were associated with the decreased frequency of Treg in PBMC, while FOXP3 gene variants were associated with affection status, but not with Treg frequency. CONCLUSION: SLE patients have impaired Treg production or maintenance, a trait strongly associated with SLE disease activity and autoantibody titers, and possibly resulting from the inability to convert FOXP3+CD25- into FOXP3+CD25+ T cells. Treg frequency is highly heritable within SLE families, with specific variants of the CTLA4 and TGFbeta genes contributing to this trait, while FOXP3 contributes to SLE through mechanisms not involving a modulation of Treg frequency. These findings establish that the genetic components in SLE pathogenesis include genes related to Treg generation or maintenance.

Carbon Monoxide Dehydrogenases.

Carbon monoxide dehydrogenases (CODHs) catalyze the reversible oxidation of CO with water to CO2, two electrons, and two protons. Two classes of CODHs exist, having evolved from different scaffolds featuring active sites built from different transition metals. The basic properties of both classes are described in this overview chapter.

X-Ray Crystallography of Carbon Monoxide Dehydrogenases.

Carbon monoxide dehydrogenases (CODHs) are central players in the biogeochemical carbon monoxide (CO) cycle and have been extensively studied from the ecological level to the structural/molecular level. Of the two types of CODHs, the oxygen-tolerant CODHs use a bimetallic [CuSMo(=O)OH] center connected to the protein via a pyranopterin cofactor, whereas the oxygen-sensitive CODHs contain a [NiFe4S4-OHx]-cluster. Despite the fact that we have a basic understanding of how both types of CODHs use distinct active sites to catalyze the oxidation of CO with water to CO2, two protons, and two electrons (a reversible reaction in the cases of the oxygen-sensitive CODHs), many questions remain unanswered, especially concerning the electronic structures of the intermediate states. Since these states will likely be only revealed by the interplay of experimental and theoretical methods, there is a need to obtain accurate descriptions of the active site architectures in various states and, consequently, a need to generate crystals with good diffraction quality and collect data at element-specific wavelengths in order to determine the identity of elements in the case of mixed states. This chapter provides a description of the general working protocols for the crystallization and structural analysis of Cu,Mo-CODH and Ni,Fe-CODH that facilitates the mechanistic investigations of these important metalloenzymes.

HTHP: a novel class of hexameric, tyrosine-coordinated heme proteins.

We have cloned, expressed, isolated and characterized a hexameric tyrosine-coordinated heme protein (HTHP) from the marine bacterium Silicibacter pomeroyi. HTHP shows peroxidase and catalase activity and has a high thermal stability. As-isolated HTHP has absorption maxima at 407, 495, 504, 532 and 622 nm wavelength. Upon reduction maxima at 430, 564 and 596 nm wavelength are discernible. The crystal structure of HTHP reveals a hexameric, ring-like arrangement of six monomers. Each monomer binds a solvent accessible heme group, which is stabilized by the interaction of three neighboring monomers. The pocket around the heme distal side is positively charged due to three conserved arginine residues in direct vicinity. The heme iron is penta-coordinated with a tyrosine residue as proximal ligand. The coordinating hydroxyl-group of the tyrosine ligand interacts with the guanidinium group of a nearby arginine residue, an arrangement closely resembling the catalytic dyad found in monofunctional heme-containing catalases and coral allene oxide synthases, which are b-type cytochromes with tyrosine coordination trans to an empty coordination site. Despite the similarity in heme coordination HTHP is functionally and structurally unrelated to catalases and other heme-containing proteins. Its hexameric arrangement, solvent accessible heme binding pocket and heme coordination by tyrosine render HTHP a unique protein with unusual properties. A database search against complete and incomplete genomes shows that the 76 amino acid residues sequence of HTHP is unrelated to characterized proteins, but is homologous to orfs found in a phylogenetically diverse set of bacteria with sequence identities of 30-76%. We therefore propose that HTHP is the prototype of a new class of heme proteins.

The role of HLA-DRB1 alleles on susceptibility and outcome of a Portuguese Multiple Sclerosis population.

BACKGROUND: The association between susceptibility to multiple sclerosis (MS) and HLA-DRB1*15 has been reported in various European populations. OBJECTIVE: To investigate the relationship between MS, HLA-DRB1*15 and other DRB1 alleles in a Portuguese population and their association with clinical course of MS. METHODS: The HLA-DRB1 alleles were analyzed by PCR-SSP in 248 MS patients and 282 healthy controls. In order to relate HLA-DRB1 alleles to disease aggressiveness, patients with relapsing remitting MS and secondary progressive MS were subdivided into 3 groups: 'benign' MS patients who maintain an Extended Disability Status Scale (EDSS) score of 3 after the same period and 'aggressive' MS those with EDSS>or=6 within 15 years of disease onset. RESULTS: As expected, a higher frequency of HLA-DRB1*15 was found in MS patients (29.8% vs 19.9%, odds ratio (OR)=1.72, 95% CI=1.15-2.56, p=0.008). The HLA-DRB1*03 allele was positively associated with MS in the overall patient population (22.6% vs 15.6%, OR=1.58, 95% CI=1.02-2.45). Concerning disease aggressiveness, HLA-DRB1*15 occurred more frequently in the group with benign disease (42.6% vs 19.9%, OR=2.99, 95% CI=1.56-5.72) and in the group with non-benign disease (34.1% vs 19.9%, OR=2.09, 95% CI=1.05-4.16) compared with controls. When time to reach an EDSS=3 or EDSS=6 was considered as end point, HLA-DRB1*15 negative patients were found to have a worse prognosis. CONCLUSIONS: In this population of Portuguese MS patients, the HLA-DRB1*15 allele is established as a genetic marker for susceptibility to MS and is also associated with a better outcome.

2-Oxoquinoline 8-monooxygenase oxygenase component: active site modulation by Rieske-[2Fe-2S] center oxidation/reduction.

2-Oxoquinoline 8-monooxygenase is a Rieske non-heme iron oxygenase that catalyzes the NADH-dependent oxidation of the N-heterocyclic aromatic compound 2-oxoquinoline to 8-hydroxy-2-oxoquinoline in the soil bacterium Pseudomonas putida 86. The crystal structure of the oxygenase component of 2-oxoquinoline 8-monooxygenase shows a ring-shaped, C3-symmetric arrangement in which the mononuclear Fe(II) ion active site of one monomer is at a distance of 13 A from the Rieske-[2Fe-2S] center of a second monomer. Structural analyses of oxidized, reduced, and substrate bound states reveal the molecular bases for a new function of Fe-S clusters. Reduction of the Rieske center modulates the mononuclear Fe through a chain of conformational changes across the subunit interface, resulting in the displacement of Fe and its histidine ligand away from the substrate binding site. This creates an additional coordination site at the mononuclear Fe(II) ion and can open a pathway for dioxygen to bind in the substrate-containing active site.