The Tetrameric Structure of Nucleotide-regulated Pyrophosphatase and Its Modulation by Deletion Mutagenesis and Ligand Binding

ABSTRACTA quarter of prokaryotic Family II inorganic pyrophosphatases (PPases) contain a regulatory insert comprised of two cystathionine β-synthase (CBS) domains and one DRTGG domain in addition to the two catalytic domains that form canonical Family II PPases. The CBS domain-containing PPases (CBS-PPases) are allosterically activated or inhibited by adenine nucleotides that cooperatively bind to the CBS domains. Here we use chemical cross-linking and analytical ultracentrifugation to show that CBS-PPases from Desulfitobacterium hafniense and four other bacterial species are active as 200–250-kDa homotetramers, which seems unprecedented among the four PPase families. The tetrameric structure is stabilized by Co2+, the essential cofactor, pyrophosphate, the substrate, and adenine nucleotides, including diadenosine tetraphosphate. The deletion variants of dhPPase containing only catalytic or regulatory domains are dimeric. Co2+ depletion by incubation with EDTA converts CBS-PPase into inactive tetrameric and dimeric forms. Dissociation of tetrameric CBS-PPase and its catalytic part by dilution renders them inactive. The structure of CBS-PPase tetramer was modelled from the structures of dimeric catalytic and regulatory parts. These findings signify the role of the unique oligomeric structure of CBS-PPase in its multifaced regulation.

Investigation of Biogenic Passivating Layers on Corroded Iron

This study evaluates mechanisms of biogenic mineral formation induced by bacterial iron reduction for the stabilization of corroded iron. As an example, the Desulfitobacterium hafniense strain TCE1 was employed to treat corroded coupons presenting urban natural atmospheric corrosion, and spectroscopic investigations were performed on the samples’ cross-sections to evaluate the corrosion stratigraphy. The treated samples presented a protective continuous layer of iron phosphates (vivianite Fe2+3(PO4)2·8H2O and barbosalite Fe2+Fe3+2(PO4)2(OH)2), which covered 92% of the surface and was associated with a decrease in the thickness of the original corrosion layer. The results allow us to better understand the conversion of reactive corrosion products into stable biogenic minerals, as well as to identify important criteria for the design of a green alternative treatment for the stabilization of corroded iron.

Sulfated Metabolites of Flavonolignans and 2,3-Dehydroflavonolignans: Preparation and Properties

Silymarin, an extract from milk thistle (Silybum marianum) fruits, is consumed in various food supplements. The metabolism of silymarin flavonolignans in mammals is complex, the exact structure of their metabolites still remains partly unclear and standards are not commercially available. This work is focused on the preparation of sulfated metabolites of silymarin flavonolignans. Sulfated flavonolignans were prepared using aryl sulfotransferase from Desulfitobacterium hafniense and p-nitrophenyl sulfate as a sulfate donor and characterized by high-resolution mass spectrometry (HRMS) and nuclear magnetic resonance (NMR). Their 1,1-diphenyl-2-picrylhydrazyl (DPPH), 2,2′-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), and N,N-dimethyl-p-phenylenediamine (DMPD) radical scavenging; ferric (FRAP) and Folin–Ciocalteu reagent (FCR) reducing activity; anti-lipoperoxidant potential; and effect on the nuclear erythroid 2-related factor 2 (Nrf2) signaling pathway were examined. Pure silybin A 20-O-sulfate, silybin B 20-O-sulfate, 2,3-dehydrosilybin-20-O-sulfate, 2,3-dehydrosilybin-7,20-di-O-sulfate, silychristin-19-O-sulfate, 2,3-dehydrosilychristin-19-O-sulfate, and silydianin-19-O-sulfate were prepared and fully characterized. Sulfated 2,3-dehydroderivatives were more active in FCR and FRAP assays than the parent compounds, and remaining sulfates were less active chemoprotectants. The sulfated flavonolignans obtained can be now used as authentic standards for in vivo metabolic experiments and for further research on their biological activity.

Interaction between microbes, iron and chlorine for the development of biotechnological approaches to stabilize corroded iron

Iron objects suffer inexorable oxidation and without any human intervention they would be completely damaged. This phenomenon occurs for iron surfaces, outdoor exposed structures as well as for archaeological iron objects. Several methods are currently available for the stabilisation of this type of metallic substrate, however, none of them is completely efficient, and several relay on the use of hazardous compounds. In addition, especially for outdoor iron and pipelines structure a permanent protective treatment does not exist. After few years these corroded surfaces have to be re-treated and in some cases replaced. This causes substantial maintenance costs having an important economic impact on our society. Regarding archaeological iron objects, an additional issue has to be considered. In fact, each object consists of a unique testimony of our past that should be preserved and studied. An archaeological object is usually unique and if the conservation interventions fail, all the information that the object could have revealed will be lost. Scientists agree with the fact that until now an efficient and durable stabilisation treatment for corroded iron does not exist. As a consequence, there is a pressing need to investigate new approaches. To this purpose, the present thesis investigated the potential of microorganisms (bacteria and fungi) for the development of stabilisation methods for corroded iron. Since one of the main issues for this metal is chlorine, this study examined two different strategies of chlorine removal and conversion of the unstable iron compounds into more stable biogenic minerals. The first approach was an indirect chlorine extraction, consisting on the microbial removal of iron ions present in chlorinated corrosion compounds. For this purpose, microbial biogenic minerals production and fungal iron adsorption were investigated. In particular, exploiting biogenic minerals production of the strains TCE1 and LBE of the anaerobic bacterium Desulfitobacterium hafniense, it was possible to convert a part of the corrosion layer of corroded iron coupons, as well as of archaeological iron nails, into biogenic vivianite and magnetite. In addition, this study allowed definitely to assess that fungi are not the best candidates to develop stabilisation methods for corroded iron based on biogenic minerals production. In fact, even though Beauveria bassiana produced some biogenic crystals their amount was not sufficient for a precise characterisation, and none of the factors tested stimulated a higher production. Nevertheless, interesting results were obtained for fungal iron uptake. Indeed, iron uptake of the fungus Alternaria sp. was successfully used for a biocleaning of corroded iron coupons. In addition, another biotechnological application exploiting fungal iron uptake was investigated. In this study the ability of bacteria to use iron chelated in fungal dead biomass as a bioavailable source of iron was proved for Pseudomonas fluorescens. This could then be exploited to improve iron bioavailability, as well as availability of organic carbon in soil for other microbes and maybe also plants. A second approach regarding a direct method for the removal of chlorine was also studied. Uptake of potassium and chlorine was proved for B. bassiana that produced aggregates containing these elements onto its biomass when exposed to FeCl2. However this ability could not be further exploited, as chlorine uptake was not the main resistance mechanism used by this fungus against chlorine, and an efficient uptake of this ion was not measured. Finally, aiming to remove chlorine from corroded iron, volatiles organic compounds production was studied. Preliminary results showed that NaCl stimulates the production of particular compounds not present in absence of this substance. Overall it can be affirmed that this study allow to assess that microorganisms are a valuable alternative for the stabilisation of corroded iron. Bacteria could be employed to stabilize the corrosion layer by producing stable biogenic minerals, while fungi could be used for biocleaning of corroded iron.

An asparagine residue mediates intramolecular communication in nucleotide-regulated pyrophosphatase

Many prokaryotic soluble PPases (pyrophosphatases) contain a pair of regulatory adenine nucleotide-binding CBS (cystathionine β-synthase) domains that act as ‘internal inhibitors’ whose effect is modulated by nucleotide binding. Although such regulatory domains are found in important enzymes and transporters, the underlying regulatory mechanism has only begun to come into focus. We reported previously that CBS domains bind nucleotides co-operatively and induce positive kinetic co-operativity (non-Michaelian behaviour) in CBS-PPases (CBS domain-containing PPases). In the present study, we demonstrate that a homodimeric ehPPase (Ethanoligenens harbinense PPase) containing an inherent mutation in an otherwise conserved asparagine residue in a loop near the active site exhibits non-co-operative hydrolysis kinetics. A similar N312S substitution in ‘co-operative’ dhPPase (Desulfitobacterium hafniense PPase) abolished kinetic co-operativity while causing only minor effects on nucleotide-binding affinity and co-operativity. However, the substitution reversed the effect of diadenosine tetraphosphate, abolishing kinetic co-operativity in wild-type dhPPase, but restoring it in the variant dhPPase. A reverse serine-to-asparagine replacement restored kinetic co-operativity in ehPPase. Molecular dynamics simulations revealed that the asparagine substitution resulted in a change in the hydrogen-bonding pattern around the asparagine residue and the subunit interface, allowing greater flexibility at the subunit interface without a marked effect on the overall structure. These findings identify this asparagine residue as lying at the ‘crossroads’ of information paths connecting catalytic and regulatory domains within a subunit and catalytic sites between subunits.

Draft Genome Sequence of Desulfitobacterium hafniense Strain DH, a Sulfate-Reducing Bacterium Isolated from Paddy Soils

Desulfitobacterium hafniense strain DH is a sulfate-reducing species. Here, we report the draft genome sequence of strain DH, with a size of 5,368,588 bp, average G+C content of 47.48%, and 5,296 predicted protein-coding sequences.

Structures of the methyltransferase component ofDesulfitobacterium hafnienseDCB-2O-demethylase shed light on methyltetrahydrofolate formation

O-Demethylation by acetogenic or organohalide-respiring bacteria leads to the formation of methyltetrahydrofolate from aromatic methyl ethers.O-Demethylases, which are cobalamin-dependent, three-component enzyme systems, catalyse methyl-group transfers from aromatic methyl ethers to tetrahydrofolateviamethylcobalamin intermediates. In this study, crystal structures of the tetrahydrofolate-binding methyltransferase module from aDesulfitobacterium hafnienseDCB-2O-demethylase were determined both in complex with tetrahydrofolate and the product methyltetrahydrofolate. While these structures are similar to previously determined methyltransferase structures, the position of key active-site residues is subtly altered. A strictly conserved Asn is displaced to establish a putative proton-transfer network between the substrate N5 and solvent. It is proposed that this supports the efficient catalysis of methyltetrahydrofolate formation, which is necessary for efficientO-demethylation.