Aristodemo Carpen

A persulfurated cysteine promotes active site reactivity in Azotobacter vinelandii rhodanese

Abstract Active site reactivity and specificity of RhdA, a thiosulfate:cyanide sulfurtransferase (rhodanese) from Azotobacter vinelandii, have been investigated through ligand binding, sitedirected mutagenesis, and Xray crystallographic techniques, in a combined approach. In native RhdA the active site Cys230 is found persulfurated; fluorescence and sulfurtransferase activity measurements show that phosphate anions interact with Cys230 persulfide sulfur atom and modulate activity. Crystallographic analyses confirm that phosphate and hypophosphite anions react with native RhdA, removing the persulfide sulfur atom from the active site pocket. Considering that RhdA and the catalytic subunit of Cdc25 phosphatases share a common threedimensional fold as well as active site Cys (catalytic) and Arg residues, two RhdA mutants carrying a single amino acid insertion at the active site loop were designed and their phosphatase activity tested. The crystallographic and functional results reported here show that specific sulfurtransferase or phosphatase activities are strictly related to precise tailoring of the catalytic loop structure in RhdA and Cdc25 phosphatase, respectively.

A simple and reliable methodology to detect egg white in art samples

A protocol for a simple and reliable dot-blot immunoassay was developed and optimized to test work of art samples for the presence of specific proteinaceus material (i.e. ovalbumin-based). The analytical protocol has been extensively set up with respect, among the other, to protein extraction conditions, to densitometric analysis and to the colorimetric reaction conditions. Feasibility evaluation demonstrated that a commercial scanner and a free image analysis software can be used for the data acquisition and elaboration, thus facilitating the application of the proposed protocol to commonly equipped laboratories and to laboratories of museums and conservation centres. The introduction of method of standard additions in the analysis of fresh and artificially aged laboratory-prepared samples, containing egg white and various pigments, allowed us to evaluate the matrix effect and the effect of sample aging and to generate threshold density values useful for the detection of ovalbumin in samples from ancient works of art. The efficacy of the developed dot-blot immunoassay was proved testing microsamples from 13th–16th century mural paintings of Saint Francesco Church in Lodi (Italy). Despite the aging, the altered conditions of conservation, the complex matrix, and the micro-size of samples, the presence of ovalbumin was detected in all those mural painting samples where mass-spectrometry-based proteomic analysis unambiguously detected ovalbumin peptides.

The lack of rhodanese RhdA affects the sensitivity of Azotobacter vinelandii to oxidative events

The rhdA gene of Azotobacter vinelandii codes for RhdA, a rhodanese-domain protein with an active-site loop structure which has not currently been found in proteins of the rhodanese-homology superfamily. Considering the lack of information on the functional role of the ubiquitous rhodaneses, in the present study we examined the in vivo functions of RhdA by using an A. vinelandii mutant strain (MV474), in which the rhdA gene was disrupted by deletion. Preliminary phenotypic characterization of the rhdA mutant suggested that RhdA could exert protection over Fe-S enzymes, which are easy targets for oxidative damage. To highlight the role of RhdA in preserving sensitive Fe-S clusters, in the present study we analysed the defects of the rhdA-null strain by exploiting growth conditions which resulted in enhancing the catalytic deficiency of enzymes with vulnerable Fe-S clusters. We found that a lack of RhdA impaired A. vinelandii growth in the presence of gluconate, a carbon source that activates the Entner-Doudoroff pathway in which the first enzyme, 6-phosphogluconate dehydratase, employs a 4Fe-4S cluster as an active-site catalyst. By combining proteomics, enzymatic profiles and model systems to generate oxidative stress, evidence is provided that to rescue the effects of a lack of RhdA, A. vinelandii needed to activate defensive activities against oxidative damage. The possible functionality of RhdA as a redox switch which helps A. vinelandii in maintaining the cellular redox balance was investigated by using an in vitro model system that demonstrated reversible chemical modifications in the highly reactive RhdA Cys(230) thiol.

Effects of the deficiency of the rhodanese-like protein RhdA in Azotobacter vinelandii

In Azotobacter vinelandii the rhdA gene codes for a protein (RhdA) of the rhodanese‐homology superfamily. By combining proteomics, enzymic profiles and ultrastructural observations, the phenotype of an A. vinelandii rhdA mutant was analyzed. We found that the A. vinelandii rhdA mutant, and not the wild‐type strain, accumulated polyhydroxybutyrate. RhdA deficiency enhanced the expression of enzymes of the polyhydroxybutyrate biosynthetic operon, and affected the activity of specific tricarboxylic acid cycle enzymes. The effect was dramatic on aconitase, in spite of comparable expression of aconitase polypeptides in both strains. By using a model system, we found that RhdA triggered protection from oxidants.

Mutagenic analysis of Thr‐232 in rhodanese from Azotobacter vinelandii highlighted the differences of this prokaryotic enzyme from the known sulfurtransferases

Azotobacter vinelandii RhdA uses thiosulfate as the only sulfur donor in vitro, and this apparent selectivity seems to be a unique property among the characterized sulfurtransferases. To investigate the basis of substrate recognition in RhdA, we replaced Thr‐232 with either Ala or Lys. Thr‐232 was the target of this study since the corresponding Lys‐249 in bovine rhodanese has been identified as necessary for catalytic sulfur transfer, and replacement of Lys‐249 with Ala fully inactivates bovine rhodanese. Both T232K and T232A mutants of RhdA showed significant increase in thiosulfate‐cyanide sulfurtransferase activity, and no detectable activity in the presence of 3‐mercaptopyruvate as the sulfur donor substrate. Fluorescence measurements showed that wild‐type and mutant RhdAs were overexpressed in the persulfurated form, thus conferring to this enzyme the potential of a persulfide sulfur donor compound. RhdA contains a unique sequence stretch around the catalytic cysteine, and the data here presented suggest a possible divergent physiological function of A. vinelandii sulfurtransferase.

Biochemical and Functional Characterization of an Albumin Protein Belonging to the Hemopexin Superfamily from Lens culinaris Seeds

The present paper reports the purification and biochemical characterization of an albumin identified in mature lentil seeds with high sequence similarity to pea PA2. These proteins are found in many edible seeds and are considered potentially detrimental for human health due to the potential allergenicity and lectin-like activity. Thus, the description of their possible presence in food and the assessment of the molecular properties are relevant. The M(r), pI, and N-terminal sequence of this protein have been determined. The work included the study of (i) the binding properties to hemine to assess the presence of hemopexin structural domains and (ii) the binding properties of the protein to thiamin. In addition, the structural changes induced by heating have been evaluated by means of spectroscopic techniques. Denaturation temperature has also been determined. The present work provides new insights about the structural molecular features and the ligand-binding properties and dynamics of this kind of seed albumin.

Macromolecular and Micronutrient Profiles of Sprouted Chickpeas to Be Used for Integrating Cereal-Based Food

Pulse flour may be used to improve nutritional traits of gluten and gluten-free formulations in traditional food such as bread or pasta. However, owing to some intrinsic nutritional, textural, and sensory properties, the use of pulses as ingredients for production of enriched food remains limited. In this study, we investigated the modification in macromolecules and micronutrients in industrial-scale flour from partially sprouted chickpeas to define its possible use as an ingredient in cereal-based foods. Controlled sprouting resulted in significant decrease of antinutritional compounds (e.g., phytic acid and serine protease inhibitors) and in an increase of free minerals and vitamins. Sprouting also affected the overall structural organization of proteins (such as aggregate formation) and their thiol/disulfide balance, and it promoted release of peptides. All of these had a positive effect on dough mixing properties, in particular for dough development. Formulations with enrichment in sprouted chickpea f...

Soybean-Enriched Snacks Based on African Rice

Snacks were produced by extruding blends of partially-defatted soybean flour with flours from milled or parboiled African-grown rice. The interplay between composition and processing in producing snacks with a satisfactory sensory profile was addressed by e-sensing, and by molecular and rheological approaches. Soybean proteins play a main role in defining the properties of the protein network in the products. At the same content in soybean flour, use of parboiled rice flour increases the snack’s hardness. Electronic nose and electronic tongue discriminated samples containing a higher amount of soybean flour from those with a lower soybean flour content.

SseA, a 3-mercaptopyruvate sulfurtransferase from Escherichia coli: crystallization and preliminary crystallographic data

SseA, the translation product of the Escherichia coli sseA gene, is a 31 kDa protein endowed with 3-mercaptopyruvate:cyanide sulfurtransferase activity in vitro. As such, SseA is the prototype of a sulfurtransferase subfamily distinguished from the better known rhodanese sulfurtransferases, which display thiosulfate:cyanide sulfurtransferase activity. The physiological role of the two homologous enzyme families, whose catalytic activity is centred on a reactive invariant cysteine, is a matter of debate. In this framework, the forthcoming crystal structure analysis of SseA will be based on the tetragonal crystal form (space group P4(1) or P4(3)) reported here, with unit-cell parameters a = b = 150.2, c = 37.9 A.

Enhanced vitamin B12 production in an innovative lupin tempeh is due to synergic effects of Rhizopus and Propionibacterium in cofermentation

Abstract Fermentation represents a valuable and cost-effective approach for food stabilisation and nutritional improvement. Tempeh is an example of soybean solid-state fermentation. In this work, we investigated the possibility of producing a tempeh analogue containing high amounts of vitamin B12 using seeds of three different species of the legume lupin, namely Lupinus albus, L. angustifolius and L. mutabilis, with Rhizopus oligosporus and Propionibacterium freudenreichii cofermentation. Synergic effects of Rhizopus and Propionibacterium in increasing vitamin B12 up to 1230 ng/g dw was observed. These findings indicate that this cofermentation can improve lupin nutritional quality and safety to provide a tempeh analogue with added value for vegan and vegetarian communities and low-income populations. The level of potentially toxic lupin alkaloids was also monitored during the tempeh preparation.