Glutaredoxin-like protein (GLP)-a novel bacteria sulfurtransferase that protects cells against cyanide and oxidative stresses.

The pathogen Xylella fastidiosa belongs to the Xanthomonadaceae family, a large group of Gram-negative bacteria that cause diseases in many economically important crops. A predicted gene, annotated as glutaredoxin-like protein (glp), was found to be highly conserved among the genomes of different genera within this family and highly expressed in X. fastidiosa. Analysis of the GLP protein sequences revealed three protein domains: one similar to monothiol glutaredoxins (Grx), an Fe-S cluster and a thiosulfate sulfurtransferase/rhodanese domain (Tst/Rho), which is generally involved in sulfur metabolism and cyanide detoxification. To characterize the biochemical properties of GLP, we expressed and purified the X. fastidiosa recombinant GLP enzyme. Grx activity and Fe-S cluster formation were not observed, while an evaluation of Tst/Rho enzymatic activity revealed that GLP can detoxify cyanide and transfer inorganic sulfur to acceptor molecules in vitro. The biological activity of GLP relies on the cysteine residues in the Grx and Tst/Rho domains (Cys33 and Cys266, respectively), and structural analysis showed that GLP and GLPC266S were able to form high molecular weight oligomers (> 600 kDa), while replacement of Cys33 with Ser destabilized the quaternary structure. In vivo heterologous enzyme expression experiments in Escherichia coli revealed that GLP can protect bacteria against high concentrations of cyanide and hydrogen peroxide. Finally, phylogenetic analysis showed that homologous glp genes are distributed across Gram-negative bacterial families with conservation of the N- to C-domain order. However, no eukaryotic organism contains this enzyme. Altogether, these results suggest that GLP is an important enzyme with cyanide-decomposing and sulfurtransferase functions in bacteria, whose presence in eukaryotes we could not observe, representing a promising biological target for new pharmaceuticals.

Rhodanese incorporated in Langmuir and Langmuir-Blodgett films of dimyristoylphosphatidic acid: Physical chemical properties and improvement of the enzyme activity.

Preserving the catalytic activity of enzymes immobilized in bioelectronics devices is essential for optimal performance in biosensors. Therefore, ultrathin films in which the architecture can be controlled at the molecular level are of interest. In this work, the enzyme rhodanese was adsorbed onto Langmuir monolayers of the phospholipid dimyristoylphosphatidic acid and characterized by surface pressure-area isotherms, polarization-modulated infrared reflection-absorption spectroscopy (PM-IRRAS), and Brewster angle microscopy (BAM). The incorporation of the enzyme (5% in mol) in the lipid monolayer expanded the film, providing small surface domains, as visualized by BAM. Also, amide bands could be identified in the PM-IRRAS spectra, confirming the presence of the enzyme at the air-water interface. Structuring of the enzyme into α-helices was identified in the mixed monolayer and was preserved when the film was transferred from the liquid interface to solids supports as Langmuir-Blodgett (LB) films. The enzyme-lipid LB films were then characterized by fluorescence spectroscopy, PM-IRRAS, and atomic force microscopy. Measurements of the catalytic activity towards cyanide showed that the enzyme accommodated in the LB films preserved more than 87% of the enzyme activity in relation to the homogeneous medium. After 1 month, the enzyme in the LB film maintained 85% of the activity in contrast to the homogeneous medium, which 24% of the enzyme activity was kept. The method presented in this work not only points to an enhanced catalytic activity toward cyanide, but also may explain why certain film architectures exhibit an improved performance.