An Electrochemical Approach to Follow and Evaluate the Kinetic Catalysis of Ricin on hsDNA

International authorities classify the ricin toxin, present in castor seeds, as a potential agent for use in bioterrorism. Therefore, the detection, identification, and characterization of ricin are considered the first actions for its risk assessment during a suspected exposure, parallel to the development of therapeutic and medical countermeasures. In this study, we report the kinetic analysis of electro-oxidation of adenine released from hsDNA by the catalytic action of ricin by square wave voltammetry. The results suggest that ricin-mediated adenine release exhibited an unusual kinetic profile, with a progress curve controlled by the accumulation of the product and the values of the kinetic constants of 46.6 µM for Km and 2000 min−1 for kcat, leading to a catalytic efficiency of 7.1 × 105 s−1 M−1.

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A Simple, Fast and Portable Method for Electrochemical Detection of Adenine Released by Ricin Enzymatic Activity

Toxins ◽

10.3390/toxins13040238 ◽

2021 ◽

Vol 13 (4) ◽

pp. 238

Author(s):

George Oliveira ◽

José Maurício Schneedorf

Keyword(s):

Linear Response ◽

Electrochemical Assay ◽

Experimental Conditions ◽

Ricin Toxin ◽

Sperm Dna ◽

Electro Oxidation ◽

Food Matrices ◽

Dna Substrate ◽

Identification And Characterization

International authorities classify ricin toxin present in castor seed as a potential agent for use in bioterrorism. Therefore, the detection, identification, and characterization of ricin in various sample matrices are considered necessary actions for risk assessment during a suspected exposure. This study reports a portable electrochemical assay for detecting active ricin based on the adenine electro-oxidation released from herring sperm DNA substrate by its catalytic action. Also, kinetic parameters were calculated, and the values were Km of 3.14 µM and Kcat 2107 min−1. A linear response was found in optimized experimental conditions for ricin concentrations ranging from 8 to 120 ng/mL, and with a detection limit of 5.14 ng/mL. This proposed detection strategy emphasizes the possibility of field detection of active ricin in food matrices and can be applied to other endonucleolytic activities.

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Postgenomic Scan of Metallo-β-Lactamase Homologues in Rhizobacteria: Identification and Characterization of BJP-1, a Subclass B3 Ortholog from Bradyrhizobium japonicum

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01551-05 ◽

2006 ◽

Vol 50 (6) ◽

pp. 1973-1981 ◽

Cited By ~ 31

Author(s):

Magdalena Stoczko ◽

Jean-Marie Frère ◽

Gian Maria Rossolini ◽

Jean-Denis Docquier

Keyword(s):

Bradyrhizobium Japonicum ◽

Catalytic Efficiency ◽

Open Reading Frames ◽

Human Pathogens ◽

Microbial Genomes ◽

E Coli ◽

Genes Encoding ◽

And Function ◽

Identification And Characterization

ABSTRACT The diffusion of metallo-β-lactamases (MBLs) among clinically important human pathogens represents a therapeutic issue of increasing importance. However, the origin of these resistance determinants is largely unknown, although an important number of proteins belonging to the MBL superfamily have been identified in microbial genomes. In this work, we analyzed the distribution and function of genes encoding MBL-like proteins in the class Rhizobiales. Among 12 released complete genomes of members of the class Rhizobiales, a total of 57 open reading frames (ORFs) were found to have the MBL conserved motif and identity scores with MBLs ranging from 8 to 40%. On the basis of the best identity scores with known MBLs, four ORFs were cloned into Escherichia coli for heterologous expression. Among their products, one (blr6230) encoded by the Bradyrhizobium japonicum USDA110 genome, named BJP-1, hydrolyzed β-lactams when expressed in E. coli. BJP-1 enzyme is most closely related to the CAU-1 enzyme from Caulobacter vibrioides (40% amino acid sequence identity), a member of subclass B3 MBLs. A kinetic analysis revealed that BJP-1 efficiently hydrolyzed most β-lactam substrates, except aztreonam, ticarcillin, and temocillin, with the highest catalytic efficiency measured with meropenem. Compared to other MBLs, BJP-1 was less sensitive to inactivation by chelating agents.

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