Catalytic Properties of Flavocytochrome c Sulfide Dehydrogenase from Haloalkaliphilic Bacterium Thioalkalivibrio paradoxus.

Flavocytochrome c sulfide dehydrogenase (FCC) is one of the central enzymes of the respiratory chain in sulfur-oxidizing bacteria. FCC catalyzes oxidation of sulfide and polysulfide ions to elemental sulfur accompanied by electron transfer to cytochrome c. The catalytically active form of the enzyme is a non-covalently linked heterodimer composed of flavin- and heme-binding subunits. The Thioalkalivibrio paradoxus ARh1 genome contains five copies of genes encoding homologous FCCs with an amino acid sequence identity from 36 to 54%. When growing on thiocyanate or thiosulfate as the main energy source, the bacterium synthesizes products of different copies of FCC genes. In this work, we isolated and characterized FCC synthesized during the growth of Tv. paradoxus on thiocyanate. FCC was shown to oxidize exclusively sulfide but not other reduced sulfur compounds, such as thiosulfate, sulfite, tetrathionate, and sulfur, and it also does not catalyze the reverse reaction of sulfur reduction to sulfide. Kinetic parameters of the sulfide oxidation reaction are characterized.

Trinuclear copper biocatalytic center forms an active site of thiocyanate dehydrogenase.

Biocatalytic copper centers are generally involved in the activation and reduction of dioxygen, with only few exceptions known. Here we report the discovery and characterization of a previously undescribed copper center that forms the active site of a copper-containing enzyme thiocyanate dehydrogenase (suggested EC 1.8.2.7) that was purified from the haloalkaliphilic sulfur-oxidizing bacterium of the genus Thioalkalivibrio ubiquitous in saline alkaline soda lakes. The copper cluster is formed by three copper ions located at the corners of a near-isosceles triangle and facilitates a direct thiocyanate conversion into cyanate, elemental sulfur, and two reducing equivalents without involvement of molecular oxygen. A molecular mechanism of catalysis is suggested based on high-resolution three-dimensional structures, electron paramagnetic resonance (EPR) spectroscopy, quantum mechanics/molecular mechanics (QM/MM) simulations, kinetic studies, and the results of site-directed mutagenesis.

Identification of Na+-Pumping Cytochrome Oxidase in the Membranes of Extremely Alkaliphilic Thioalkalivibrio Bacteria.

For the first time, the functioning of the oxygen reductase Na+-pump (Na+-pumping cytochrome c oxidase of the cbb3-type) was demonstrated by examining the respiratory chain of the extremely alkaliphilic bacterium Thioalkalivibrio versutus [Muntyan, M. S., et al. (2015) Cytochrome cbb3 of Thioalkalivibrio is a Na+-pumping cytochrome oxidase, Proc. Natl. Acad. Sci. USA, 112, 7695-7700], a product of the ccoNOQP operon. In this study, we detected and identified this enzyme using rabbit polyclonal antibody against the predicted C-terminal amino acid sequence of its catalytic subunit. We found that this cbb3-type oxidase is synthesized in bacterial cells, where it is located in the membranes. The 48-kDa oxidase subunit (CcoN) is catalytic, while subunits CcoO and CcoP with molecular masses of 29 and 34 kDa, respectively, are cytochromes c. The theoretical pI values of the CcoN, CcoO, and CcoP subunits were determined. It was shown that parts of the CcoO and CcoP subunits exposed to the aqueous phase on the cytoplasmic membrane P-side are enriched with negatively charged amino acid residues, in contrast to the parts of the integral subunit CcoN adjacent to the aqueous phase. Thus, the Na+-pumping cytochrome c oxidase of T. versutus, both in function and in structure, demonstrates adaptation to extremely alkaline conditions.

The LH2 Complexes (B800-850 and B800-830) Are Assembled in the Cells of the Sulfur Bacterium Thiorhodospira sibirica Strain Kir-3 without Carotenoids.

The results of assembling the light-harvesting complexes in the cells of the purple sulfur bacterium Thiorhodospira (T.) sibirica strain Kir-3 while suppressing the biosynthesis of carotenoids with diphenylamine (DPA) were studied. LH2 complexes (B800-850 and B800-830) with different carotenoid composition were isolated from the cells obtained. Maximum inhibition of carotenoid biosynthesis (~90% of the control) was reached at an inhibitor concentration of 53.25 µM (9 mg/L). It was established that changes in the qualitative and quantitative composition of carotenoids do not affect the assembly of B800-830 and B800-850 complexes. It is assumed that, in the population of DPA-LH2 complexes from T. sibirica strain Kir-3, both the carotenoidless complexes and the complexes containing one or two carotenoid molecules can be assembled. These results support the hypothesis that carotenoids are not required for assembling B800-850 and B800-830 complexes.

Covalent immobilization of a halophilic, alkalithermostable lipase LipR2 on Florisil® nanoparticles for production of alkyl levulinates.

A novel halophilic, alkalithermostable lipase LipR2 from Alkalispirillum sp. NM-ROO2 was cloned and expressed. LipR2 was covalently immobilized on Florisil® functionalized with glutaraldehyde. Protein binding efficiency of functionalized Florisil® was 94.7%. Immobilized LipR2 retained 97.5% of specific activity of the free enzyme. Free LipR2 has maximal activity at 52 °C, pH 9.3 and 1.9 M NaCl and is resistant to surfactants and organic solvents. Immobilization enhanced LipR2's extreme characteristics, and increased thermostability of LipR2 with the half-life at 50 °C increasing three-fold. Immobilized LipR2 was used as a biocatalyst for esterification of levulinic acid with n-butanol. Under optimal conditions, a 45.9% ester yield was obtained after 12 h. Immobilized LipR2 catalyzed production of ethyl levulinate and 1-dodecyl levulinate with 48.8% and 26.2% ester yields, respectively. When used in repetitive batch esterification, LipR2 retained 69%, 57% and 18.5% of initial activity on esterification of levulinic acid with ethanol, n-butanol and 1-dodecanol, respectively.

[Peroxidase activity of octaheme nitrite reductases from bacteria of the Thioalkalivibrio genus].

Closely related penta- and octaheme nitrite reductases catalyze the reduction of nitrite, nitric oxide, and hydroxylamine to ammonium and of sulfite to sulfide. NrfA pentaheme nitrite reductase plays the key role in anaerobic nitrate respiration and the protection of bacterial cells from stresses caused by nitrogen oxides and hydrogen peroxide. Octaheme nitrite reductases from bacteria of the Thioalkalivibrio genus are less studied, and their function in the cell is unknown. In order to estimate the possible role of octaheme nitrite reductases in the cell resistance to oxidative stress, the peroxidase activity of the enzyme from T. nitratireducens (TvNiR) has been studied in detail. Comparative analysis of the active site structure of TvNiR and cytochrome c peroxidases has shown some common features, such as a five-coordinated catalytic heme and identical catalytic residues in active sites. A model of the possible productive binding of peroxide at the active site of TvNiR has been proposed. The peroxidase activity has been measured for TvNiR hexamers and trimers under different conditions (pH, buffers, the addition of CaCl2 and EDTA). The maximum peroxidase activity of TvNiR with ABTS as a substrate (k cat = 17 s­1; k cat/K m = 855 mM­1 s­1) has been 100­300 times lower than the activity of natural peroxidases. The different activities of TvNiR trimers and hexamers indicate that the rate-limiting stage of the reaction is not the catalytic event at the active site but the electron transfer along the heme c electron-transport chain.

Characterization of a novel high-pH-tolerant laccase-like multicopper oxidase and its sequence diversity in Thioalkalivibrio sp.

Laccases are oxidoreductases mostly studied in fungi, while bacterial laccases remain poorly studied despite their high genetic diversity and potential for biotechnological application. Our previous bioinformatic analysis identified alkaliphilic bacterial strains Thioalkalivibrio sp. as potential sources of robust bacterial laccases that would be stable at high pH. In the present work, a gene for a laccase-like enzyme from Thioalkalivibrio sp. ALRh was cloned and expressed as a 6× His-tagged protein in Escherichia coli. The purified enzyme was a pH-tolerant laccase stable in the pH range between 2.1 and 9.9 at 20 °C as shown by intrinsic fluorescence emission spectrometry. It had optimal activities at pH 5.0 and pH 9.5 with the laccase substrates 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) and 2,6-dimethoxyphenol, respectively. In addition, it could oxidize several other monophenolic compounds and potassium hexacyanoferrate(II) but not tyrosine. It showed highest activity at 50 °C, making it suitable for prolonged incubations at this temperature. The present study shows that Thioalkalivibrio sp. encodes an active, alkaliphilic, and thermo-tolerant laccase and contributes to our understanding of the versatility of bacterial laccase-like multicopper oxidases in general.

Structural study of the X-ray-induced enzymatic reaction of octahaem cytochrome C nitrite reductase.

Octahaem cytochrome c nitrite reductase from the bacterium Thioalkalivibrio nitratireducens catalyzes the reduction of nitrite to ammonium and of sulfite to sulfide. The reducing properties of X-ray radiation and the high quality of the enzyme crystals allow study of the catalytic reaction of cytochrome c nitrite reductase directly in a crystal of the enzyme, with the reaction being induced by X-rays. Series of diffraction data sets with increasing absorbed dose were collected from crystals of the free form of the enzyme and its complexes with nitrite and sulfite. The corresponding structures revealed gradual changes associated with the reduction of the catalytic haems by X-rays. In the case of the nitrite complex the conversion of the nitrite ions bound in the active sites to NO species was observed, which is the beginning of the catalytic reaction. For the free form, an increase in the distance between the oxygen ligand bound to the catalytic haem and the iron ion of the haem took place. In the case of the sulfite complex no enzymatic reaction was detected, but there were changes in the arrangement of the active-site water molecules that were presumably associated with a change in the protonation state of the sulfite ions.

Structure of a prokaryotic sodium channel pore reveals essential gating elements and an outer ion binding site common to eukaryotic channels.

Voltage-gated sodium channels (NaVs) are central elements of cellular excitation. Notwithstanding advances from recent bacterial NaV (BacNaV) structures, key questions about gating and ion selectivity remain. Here, we present a closed conformation of NaVAe1p, a pore-only BacNaV derived from NaVAe1, a BacNaV from the arsenite oxidizer Alkalilimnicola ehrlichei found in Mono Lake, California, that provides insight into both fundamental properties. The structure reveals a pore domain in which the pore-lining S6 helix connects to a helical cytoplasmic tail. Electrophysiological studies of full-length BacNaVs show that two elements defined by the NaVAe1p structure, an S6 activation gate position and the cytoplasmic tail "neck", are central to BacNaV gating. The structure also reveals the selectivity filter ion entry site, termed the "outer ion" site. Comparison with mammalian voltage-gated calcium channel (CaV) selectivity filters, together with functional studies, shows that this site forms a previously unknown determinant of CaV high-affinity calcium binding. Our findings underscore commonalities between BacNaVs and eukaryotic voltage-gated channels and provide a framework for understanding gating and ion permeation in this superfamily.

Contrasting catalytic profiles of multiheme nitrite reductases containing CxxCK heme-binding motifs.

The multiheme cytochromes from Thioalkalivibrio nitratireducens (TvNiR) and Escherichia coli (EcNrfA) reduce nitrite to ammonium. Both enzymes contain His/His-ligated hemes to deliver electrons to their active sites, where a Lys-ligated heme has a distal pocket containing a catalytic triad of His, Tyr, and Arg residues. Protein-film electrochemistry reveals significant differences in the catalytic properties of these enzymes. TvNiR, but not EcNrfA, requires reductive activation. Spectroelectrochemistry implicates reduction of His/His-ligated heme(s) as being key to this process, which restricts the rate of hydroxide binding to the ferric form of the active-site heme. The K M describing nitrite reduction by EcNrfA varies with pH in a sigmoidal manner that is consistent with its modulation by (de)protonation of a residue with pK a ≈ 7.6. This residue is proposed to be the catalytic His in the distal pocket. By contrast, the K M for nitrite reduction by TvNiR decreases approximately linearly with increase of pH such that different features of the mechanism define this parameter for TvNiR. In other regards the catalytic properties of TvNiR and EcNrfA are similar, namely, the pH dependence of V max and the nitrite dependence of the catalytic current-potential profiles resolved by cyclic voltammetry, such that the determinants of these properties appear to be conserved.

Comparative structural and functional analysis of two octaheme nitrite reductases from closely related Thioalkalivibrio species.

UNLABELLED: Octaheme nitrite reductase from the haloalkaliphilic bacterium Thioalkalivibrio paradoxus was isolated and characterized. A comparative structural and functional analysis of two homologous octaheme nitrite reductases from closely related Thioalkalivibrio species was performed. It was shown that both enzymes have similar catalytic properties, owing to high structural similarity. Both enzymes are characterized by specific structural features distinguishing them from pentaheme cytochrome c nitrite reductases, such as the Tyr-Cys bond in the active site, the hexameric structure resulting in the formation of a void space inside the hexamer, and the product channel that opens into the void interior space of the hexamer. It is suggested that these specific structural features are responsible for the higher nitrite reductase activity, the greater preference for nitrite than for sulfite as a substrate, and the wider pH range of the catalytic activity of octaheme nitrite reductases than of pentaheme homologs. DATABASE: Nucleotide sequence data are available in the GenBank database under the accession number HQ665012.1. Structural data are available in the RCSB Protein Data Bank database under the accession numbers 3SXQ and 3TTB STRUCTURED DIGITAL ABSTRACT: TvPaR and TvPaR bind by x-ray crystallography (View interaction).

Covalent modifications of the catalytic tyrosine in octahaem cytochrome c nitrite reductase and their effect on the enzyme activity.

Octahaem cytochrome c nitrite reductase from Thioalkalivibrio nitratireducens (TvNiR), like the previously characterized pentahaem nitrite reductases (NrfAs), catalyzes the six-electron reductions of nitrite to ammonia and of sulfite to sulfide. The active site of both TvNiR and NrfAs is formed by the lysine-coordinated haem and His, Tyr and Arg residues. The distinguishing structural feature of TvNiR is the presence of a covalent bond between the CE2 atom of the catalytic Tyr303 and the S atom of Cys305, which might be responsible for the higher nitrite reductase activity of TvNiR compared with NrfAs. In the present study, a new modified form of the enzyme (TvNiRb) that contains an additional covalent bond between Tyr303 CE1 and Gln360 CG is reported. Structures of TvNiRb in complexes with phosphate (1.45 Šresolution) and sulfite (1.8 Šresolution), the structure of TvNiR in a complex with nitrite (1.83 Šresolution) and several additional structures were determined. The formation of the second covalent bond by Tyr303 leads to a decrease in both the nitrite and sulfite reductase activities of the enzyme. Tyr303 is located at the exit from the putative proton-transport channel to the active site, which is absent in NrfAs. This is an additional argument in favour of the involvement of Tyr303 as a proton donor in catalysis. The changes in the activity of cytochrome c nitrite reductases owing to the formation of Tyr-Cys and Tyr-Gln bonds may be associated with changes in the pK(a) value of the catalytic tyrosine.

Acidiferrobacter thiooxydans, gen. nov. sp. nov.; an acidophilic, thermo-tolerant, facultatively anaerobic iron- and sulfur-oxidizer of the family Ectothiorhodospiraceae.

A comprehensive physiological and phylogenetic characterisation was carried out of "Thiobacillus ferrooxidans" m-1, an acidophilic iron-oxidizing bacterium first described over 25 years ago. Phylogenetically, strain m-1 is a gammaproteobacterium, most closely related to alkaliphilic Ectothiorhodospira spp. and only distantly to iron-oxidizing acidithiobacilli. Physiological examination confirmed that strain m-1 can grow autotrophically not only by ferrous iron oxidation but also, in contrast to previous reports, by oxidation of elemental sulfur, sulfide and tetrathionate, using either oxygen or ferric iron as terminal electron acceptor. The bacterium was also found to be thermo-tolerant, growing optimally at 38°C and up to a maximum of 47°C. Growth in liquid media required an external osmotic potential of >2 bar, and was optimal at ~5 bar, though no growth occurred where the medium osmotic potential was close to that of sea water (~26 bar). From this, it was concluded that strain m-1 is a moderate osmophile. Strain m-1 was also shown to be diazotrophic and tolerant of elevated concentrations of many metals typically found in mine-impacted environments. On the basis of these data, m-1 is proposed as the type strain of a new genus and species of bacteria, Acidiferrobacter thiooxydans (DSM 2392, JCM 17358).

Isolation and characterization of nitrate reductase from the halophilic sulfur-oxidizing bacterium Thioalkalivibrio nitratireducens.

A novel nitrate reductase (NR) was isolated from cell extract of the haloalkaliphilic bacterium Thioalkalivibrio nitratireducens strain ALEN 2 and characterized. This enzyme is a classical nitrate reductase containing molybdopterin cofactor in the active site and at least one iron-sulfur cluster per subunit. Mass spectrometric analysis showed high homology of NR with the catalytic subunit NarG of the membrane nitrate reductase from the moderately halophilic bacterium Halomonas halodenitrificans. In solution, NR exists as a monomer with a molecular weight of 130-140 kDa and as a homotetramer of about 600 kDa. The specific nitrate reductase activity of NR is 12 micromol/min per mg protein, the maximal values being observed within the neutral range of pH. Like other membrane nitrate reductases, NR reduces chlorate and is inhibited by azide and cyanide. It exhibits a higher thermal stability than most mesophilic enzymes.

Preliminary X-ray analysis of twinned crystals of sarcosine dimethylglycine methyltransferase from Halorhodospira halochoris.

Sarcosine dimethylglycine methyltransferase (EC 2.1.1.157) is an enzyme from the extremely halophilic anaerobic bacterium Halorhodospira halochoris. This enzyme catalyzes the twofold methylation of sarcosine to betaine, with S-adenosylmethionine (AdoMet) as the methyl-group donor. This study presents the crystallization and preliminary X-ray analysis of recombinant sarcosine dimethylglycine methyltransferase produced in Escherichia coli. Mass spectroscopy was used to determine the purity and homogeneity of the enzyme material. Two different crystal forms, which initially appeared to be hexagonal and tetragonal, were obtained. However, on analyzing the diffraction data it was discovered that both crystal forms were pseudo-merohedrally twinned. The true crystal systems were monoclinic and orthorhombic. The monoclinic crystal diffracted to a maximum of 2.15 A resolution and the orthorhombic crystal diffracted to 1.8 A resolution.

High-resolution structural analysis of a novel octaheme cytochrome c nitrite reductase from the haloalkaliphilic bacterium Thioalkalivibrio nitratireducens.

Bacterial pentaheme cytochrome c nitrite reductases (NrfAs) are key enzymes involved in the terminal step of dissimilatory nitrite reduction of the nitrogen cycle. Their structure and functions are well studied. Recently, a novel octaheme cytochrome c nitrite reductase (TvNiR) has been isolated from the haloalkaliphilic bacterium Thioalkalivibrio nitratireducens. Here we present high-resolution crystal structures of the apoenzyme and its complexes with the substrate (nitrite) and the inhibitor (azide). Both in the crystalline state and in solution, TvNiR exists as a stable hexamer containing 48 hemes-the largest number of hemes accommodated within one protein molecule known to date. The subunit of TvNiR consists of two domains. The N-terminal domain has a unique fold and contains three hemes. The catalytic C-terminal domain hosts the remaining five hemes, their arrangement, including the catalytic heme, being identical to that found in NrfAs. The complete set of eight hemes forms a spatial pattern characteristic of other multiheme proteins, including structurally characterized octaheme cytochromes. The catalytic machinery of TvNiR resembles that of NrfAs. It comprises the lysine residue at the proximal position of the catalytic heme, the catalytic triad of tyrosine, histidine, and arginine at the distal side, channels for the substrate and product transport with a characteristic gradient of electrostatic potential, and, finally, two conserved Ca(2+)-binding sites. However, TvNiR has a number of special structural features, including a covalent bond between the catalytic tyrosine and the adjacent cysteine and the unusual topography of the product channels that open into the void interior space of the protein hexamer. The role of these characteristic structural features in the catalysis by this enzyme is discussed.

Respiratory arsenate reductase as a bidirectional enzyme.

The haloalkaliphilic bacterium Alkalilimnicola ehrlichii is capable of anaerobic chemolithoautotrophic growth by coupling the oxidation of arsenite (As(III)) to the reduction of nitrate and carbon dioxide. Analysis of its complete genome indicates that it lacks a conventional arsenite oxidase (Aox), but instead possesses two operons that each encode a putative respiratory arsenate reductase (Arr). Here we show that one homolog is expressed under chemolithoautotrophic conditions and exhibits both arsenite oxidase and arsenate reductase activity. We also demonstrate that Arr from two arsenate respiring bacteria, Alkaliphilus oremlandii and Shewanella sp. strain ANA-3, is also biochemically reversible. Thus Arr can function as a reductase or oxidase. Its physiological role in a specific organism, however, may depend on the electron potentials of the molybdenum center and [Fe-S] clusters, additional subunits, or constitution of the electron transfer chain. This versatility further underscores the ubiquity and antiquity of microbial arsenic metabolism.

Isolation and oligomeric composition of cytochrome c nitrite reductase from the haloalkaliphilic bacterium Thioalkalivibrio nitratireducens.

A new procedure for isolation of cytochrome c nitrite reductase from the haloalkaliphilic bacterium Thioalkalivibrio nitratireducens increasing significantly the yield of the purified enzyme is presented. The enzyme is isolated from the soluble fraction of the cell extract as a hexamer, as shown by gel filtration chromatography and small angle X-ray scattering analysis. Thermostability of the hexameric form of the nitrite reductase is characterized in terms of thermoinactivation and thermodenaturation.

Crystallization and preliminary X-ray analysis of cytochrome c nitrite reductase from Thioalkalivibrio nitratireducens.

A novel cytochrome c nitrite reductase (TvNiR) was isolated from the haloalkalophilic bacterium Thioalkalivibrio nitratireducens. The enzyme catalyses nitrite and hydroxylamine reduction, with ammonia as the only product of both reactions. It consists of 525 amino-acid residues and contains eight haems c. TvNiR crystals were grown by the hanging-drop vapour-diffusion technique. The crystals display cubic symmetry and belong to space group P2(1)3, with unit-cell parameter a = 194 A. A native data set was obtained to 1.5 A resolution. The structure was solved by the SAD technique using the data collected at the Fe absorption peak wavelength.