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Abstract The GPAT4 gene is considered as a potential functional candidate for single nucleotide polymorphism (SNP) studies in dairy cattle breeding due to its association with dairy performance in cattle by encoding an enzyme responsible for the presence of diacylglycerols and triacylglycerols in milk. Using the example of the GPAT4 gene, we applied the minigene splicing assay to analyze the functional consequences of its variant that was predicted to affect normal splicing. The results of functional analysis revealed the sequence variations (rs442541537), transfection experiments in a wild type and mutant cell line model system demonstrated that the investigated mutation in the second intron of the GPAT4 gene was responsible for the presence of a second exon in mature messenger RNA (mRNA). The cases of its absence in the spliced mature mRNA transcript resulted in a truncated dysfunctional protein due to the appearance of a stop codon. Thus, the discovered SNP led to alternative splicing in pre-mRNA by the 'cassette exon’ ('exon skipping’) mechanism. The studied mutation can potentially be a molecular genetic marker for alternative splicing for the GPAT4 gene and, therefore contributes to economic benefits in cattle breeding programs. [ABSTRACT FROM AUTHOR]

The optimization of all constituent conditions to obtain high and even maximum yields is a recent trend in agriculture. Legumes play a special role in this process, as they have unique characteristics with respect to storing protein and many other important components in their seeds that are useful for human and animal nutrition as well as industry and agriculture. A great advantage of legumes is the nitrogen fixation activity of their symbiotic nodule bacteria. This nitrogen self-sufficiency contributes directly to the challenging issue of feeding the world's growing population. Molybdenum is one of the most sought-after nutrients because it provides optimal conditions for the maximum efficiency of the enzymes involved in nitrogen assimilation as well as other molybdenum-containing enzymes in the host plant and symbiotic nodule bacteria. In this review, we consider the most optimal way of providing legume plants with molybdenum, its distribution in ontogeny throughout the plant, and its accumulation at the end of the growing season in the seeds. Overall, molybdenum supply improves seed quality and allows for the efficient use of the micronutrient by molybdenum-containing enzymes in the plant and subsequently the nodules at the initial stages of growth after germination. A sufficient supply of molybdenum avoids competition for this trace element between nitrogenase and nodule nitrate reductase, which enhances the supply of nitrogen to the plant. Finally, we also consider the possibility of regulating molybdenum homeostasis using modern genetic approaches. [ABSTRACT FROM AUTHOR]

Abstract: Adenylate kinase (AK) mediates the reversible transfer of phosphate groups between the adenylate nucleotides and contributes to the maintenance of their constant cellular level, necessary for energy metabolism and nucleic acid synthesis. The AK were purified from crude extracts of two sulfate-reducing bacteria (SRB), Desulfovibrio (D.) gigas NCIB 9332 and Desulfovibrio desulfuricans ATCC 27774, and biochemically and spectroscopically characterised in the native and fully cobalt- or zinc-substituted forms. These are the first reported adenylate kinases that bind either zinc or cobalt and are related to the subgroup of metal-containing AK found, in most cases, in Gram-positive bacteria. The electronic absorption spectrum is consistent with tetrahedral coordinated cobalt, predominantly via sulfur ligands, and is supported by EPR. The involvement of three cysteines in cobalt or zinc coordination was confirmed by chemical methods. Extended X-ray absorption fine structure (EXAFS) indicate that cobalt or zinc are bound by three cysteine residues and one histidine in the metal-binding site of the “LID” domain. The sequence 129Cys-X5-His-X15-Cys-X2-Cys of the AK from D. gigas is involved in metal coordination and represents a new type of binding motif that differs from other known zinc-binding sites of AK. Cobalt and zinc play a structural role in stabilizing the LID domain. [Copyright &y& Elsevier]

A novel adenylate kinase (AK) has recently been purified from Desulfovibrio gigas and characterized as a Co2+/Zn2+-containing enzyme: this is an unusual characteristic for AKs from Gram-negative bacteria, in which these enzymes are normally devoid of metals. Here, we studied the conformational stability of holo- and apo-AK as a function of temperature by differential scanning calorimetry (DSC), circular dichroism (CD), and intrinsic fluorescence spectroscopy. The thermal unfolding of AK is a cooperative two-state process, and is sufficiently reversible in the 9–11 pH range, that can be correctly interpreted in terms of a simple two-state thermodynamic model. The spectral parameters as monitored by ellipticity changes in the CD spectra of the enzyme as well as the decrease in tryptophan intensity emission upon heating were seen to be good complements to the highly sensitive but integral DSC-method. [Copyright &y& Elsevier]

Provides information on a study which showed that adenosine triphosphate sulfurylase catalyzes the formation of adenosine 5'-phosphosulfate from adenoise triphosphate and sulfate. Theoretical background; Materials and methods; Results and discussion.

Little is known of the bacterial communities associated with the rhizosphere of wild plant species found in natural settings. The rhizosphere bacterial community associated with wild thyme, Thymus zygis L., plants was analyzed using cultivation, the creation of a near-full length 16S rRNA gene clone library and 454 amplicon pyrosequencing. The bacterial community was dominated by Proteobacteria (mostly Alphaproteobacteria and Betaproteobacteria), Actinobacteria, Acidobacteria, and Gemmatimonadetes. Although each approach gave a different perspective of the bacterial community, all classes/subclasses detected in the clone library and the cultured bacteria could be found in the pyrosequencing datasets. However, an exception caused by inconclusive taxonomic identification as a consequence of the short read length of pyrotags together with the detection of singleton sequences which corresponded to bacterial strains cultivated from the same sample highlight limitations and considerations which should be taken into account when analysing and interpreting amplicon datasets. Amplicon pyrosequencing of replicate rhizosphere soil samples taken a year later permit the definition of the core microbiome associated with Thymus zygis plants. Abundant bacterial families and predicted functional profiles of the core microbiome suggest that the main drivers of the bacterial community in the Thymus zygis rhizosphere are related to the nutrients originating from the plant root and to their participation in biogeochemical cycles thereby creating an intricate relationship with this aromatic plant to allow for a feedback ecological benefit. [ABSTRACT FROM AUTHOR]

Abstract: The steady state kinetic mechanism of the H2O2-supported oxidation of different organic substrates by peroxidase from leaves of Chamaerops excelsa palm trees (CEP) has been investigated. An analysis of the initial rates vs. H2O2 and reducing substrate concentrations is consistent with a substrate-inhibited Ping-Pong Bi Bi reaction mechanism. The phenomenological approach expresses the peroxidase Ping-Pong mechanism in the form of the Michaelis–Menten equation and leads to an interpretation of the effects in terms of the kinetic parameters and of the microscopic rate constants k 1 and k 3 of the shared three-step catalytic cycle of peroxidases. [Copyright &y& Elsevier]

The industrial revolution, the first agricultural ‘green revolution’, and the development of antibiotics and therapeutic chemicals have brought significant and undeniable benefits to the human race. However, these advances demand high levels of energy, exploit natural resources and create large amounts of waste that creates an environmental burden for our planet. The pollution rate and character of many of the pollutants results in a rapid deterioration of the environment. Bioremediation functions to isolate and select microorganisms that operate under aerobic and anoxic conditions to remove these harmful pollutants. Current ‘omics’ technologies allow the exploitation of the catabolic potential of microbes without the need to cultivate them. Synthetic microbiology builds new catabolic pathways to remove recalcitrant pollutants from the environment. [ABSTRACT FROM AUTHOR]

Abstract: Royal palm tree peroxidase (RPTP) is a very stable enzyme in regards to acidity, temperature, H2O2, and organic solvents. Thus, RPTP is a promising candidate for developing H2O2-sensitive biosensors for diverse applications in industry and analytical chemistry. RPTP belongs to the family of class III secretory plant peroxidases, which include horseradish peroxidase isozyme C, soybean and peanut peroxidases. Here we report the X-ray structure of native RPTP isolated from royal palm tree (Roystonea regia) refined to a resolution of 1.85Å. RPTP has the same overall folding pattern of the plant peroxidase superfamily, and it contains one heme group and two calcium-binding sites in similar locations. The three-dimensional structure of RPTP was solved for a hydroperoxide complex state, and it revealed a bound 2-(N-morpholino) ethanesulfonic acid molecule (MES) positioned at a putative substrate-binding secondary site. Nine N-glycosylation sites are clearly defined in the RPTP electron-density maps, revealing for the first time conformations of the glycan chains of this highly glycosylated enzyme. Furthermore, statistical coupling analysis (SCA) of the plant peroxidase superfamily was performed. This sequence-based method identified a set of evolutionarily conserved sites that mapped to regions surrounding the heme prosthetic group. The SCA matrix also predicted a set of energetically coupled residues that are involved in the maintenance of the structural folding of plant peroxidases. The combination of crystallographic data and SCA analysis provides information about the key structural elements that could contribute to explaining the unique stability of RPTP. [Copyright &y& Elsevier]

Abstract: Adenylate kinase (AK) from the sulphate-reducing bacterium Desulfovibrio gigas (AK) has been characterized earlier as a Co2+/Zn2+-containing enzyme, which is an unusual characteristic for adenylate kinases from Gram-negative bacteria, in which these enzymes are normally devoid of metal ions. AK was overexpressed in E. coli and homogeneous Co2+-, Zn2+- and Fe2+-forms of the enzyme were obtained under in vivo conditions. Their structural stability and spectroscopic and kinetic properties were compared. The thermal denaturation of Co2+- and Zn2+-forms of AK was studied as a cooperative two-state process, sufficiently reversible at pH 10, which can be correctly interpreted in terms of a simple two-state thermodynamic model. In contrast, the thermally induced denaturation of Fe2+-AK is irreversible and strongly dependent upon the scan rate, suggesting that this process is under kinetic control. Practically identical contents of secondary-structure elements were found for all the metal-chelated-forms of AK upon analysis of circular dichroism data, while their tertiary structures were significantly different. The peculiar tertiary structure of Fe2+-AK, in contrast to Co2+- and Zn2+-AK, and the consequent changes in the physico-chemical and enzymatic properties of the enzyme are discussed. [Copyright &y& Elsevier]

Abstract: The structural stability of a peroxidase, a dimeric protein from palm tree Chamaerops excelsa leaves (CEP), has been characterized by high-sensitivity differential scanning calorimetry, circular dichroism and steady-state tryptophan fluorescence at pH 3. The thermally induced denaturation of CEP at this pH value is irreversible and strongly dependent upon the scan rate, suggesting that this process is under kinetic control. Moreover, thermally induced transitions at this pH value are dependent on the protein concentration, leading to the conclusion that in solution CEP behaves as dimer, which undergoes thermal denaturation coupled with dissociation. Analysis of the kinetic parameters of CEP denaturation at pH 3 was accomplished on the basis of the simple kinetic scheme , where k is a first-order kinetic constant that changes with temperature, as given by the Arrhenius equation; N is the native state, and D is the denatured state, and thermodynamic information was obtained by extrapolation of the kinetic transition parameters to an infinite heating rate. [Copyright &y& Elsevier]

Abstract: The structural stability of a peroxidase, a dimeric protein from royal palm tree (Roystonea regia) leaves, has been characterized by high-sensitivity differential scanning calorimetry, circular dichroism, steady-state tryptophan fluorescence and analytical ultracentifugation under different solvent conditions. It is shown that the thermal and chemical (using guanidine hydrochloride (Gdn-HCl)) folding/unfolding of royal palm tree peroxidase (RPTP) at pH 7 is a reversible process involving a highly cooperative transition between the folded dimer and unfolded monomers, with a free stabilization energy of about 23kcal per mol of monomer at 25°C. The structural stability of RPTP is pH-dependent. At pH 3, where ion pairs have disappeared due to protonation, the thermally induced denaturation of RPTP is irreversible and strongly dependent upon the scan rate, suggesting that this process is under kinetic control. Moreover, thermally induced transitions at this pH value are dependent on the protein concentration, allowing it to be concluded that in solution RPTP behaves as dimer, which undergoes thermal denaturation coupled with dissociation. Analysis of the kinetic parameters of RPTP denaturation at pH 3 was accomplished on the basis of the simple kinetic scheme , where k is a first-order kinetic constant that changes with temperature, as given by the Arrhenius equation; N is the native state, and D is the denatured state, and thermodynamic information was obtained by extrapolation of the kinetic transition parameters to an infinite heating rate. Obtained in this way, the value of RPTP stability at 25°C is ca. 8kcal per mole of monomer lower than at pH 7. In all probability, this quantity reflects the contribution of ion pair interactions to the structural stability of RPTP. From a comparison of the stability of RPTP with other plant peroxidases it is proposed that one of the main factors responsible for the unusually high stability of RPTP which enhances its potential use for biotechnological purposes, is its dimerization. [Copyright &y& Elsevier]

Nitrate reductases are enzymes that catalyze the conversion of nitrate to nitrite. We report here electron paramagnetic resonance (EPR) studies in the periplasmic nitrate reductase isolated from the sulfate-reducing bacteria Desulfovibrio desulfuricans ATCC 27774. This protein, belonging to the dimethyl sulfoxide reductase family of mononuclear Mo-containing enzymes, comprises a single 80-kDa subunit and contains a Mo bis(molybdopterin guanosine dinucleotide) cofactor and a [4Fe–4S] cluster. EPR-monitored redox titrations, carried out with and without nitrate in the potential range from 200 to −500 mV, and EPR studies of the enzyme, in both catalytic and inhibited conditions, reveal distinct types of Mo(V) EPR-active species, which indicates that the Mo site presents high coordination flexibility. These studies show that nitrate modulates the redox properties of the Mo active site, but not those of the [4Fe–4S] center. The possible structures and the role in catalysis of the distinct Mo(V) species detected by EPR are discussed. [ABSTRACT FROM AUTHOR]

Desulfovibrio gigas formate dehydrogenase is the first representative of a tungsten-containing enzyme from a mesophile that has been structurally characterized. It is a heterodimer of 110 and 24 kDa subunits. The large subunit, homologous to E. coli FDH-H and to D. desulfuricans nitrate reductase, harbors the W site and one [4Fe-4S] center. No small subunit ortholog containing three [4Fe-4S] clusters has been reported. The structural homology with E. coli FDH-H shows that the essential residues (SeCys158, His159, and Arg407) at the active site are conserved. The active site is accessible via a positively charged tunnel, while product release may be facilitated, for H+ by buried waters and protonable amino acids and for CO2 through a hydrophobic channel. [Copyright &y& Elsevier]