The Role of a Potential Glyoxalsase Protein in Clostridium Difficile 630

Date of Award


Degree Type


Degree Name

Master of Science in Biomedical Sciences


Basic Sciences

First Advisor

Francis E. Jenney, Jr, PhD

Second Advisor

Bonnie A Buxton, PhD

Third Advisor

Brian M Matayoshi, PhD


Clostridium difficile infection is associated with resistance to broad spectrum antibiotic therapy and is the most common cause of nosocomial infections causing pseudomembranous colitis. In addition to tolerance to antibiotic exposure, C. difficile is also able to withstand other environmental stresses, such as oxidative stress. C. difficile responds to oxidative stress by regulating expression of many genes, including some of yet unknown function such as a putative glyoxalase gene. Glyoxalase is a virtually ubiquitous system composed of two enzymes thought to be involved in oxidative stress metabolism. Glyoxalase I is a metal-containing protein involved in the conversion of methylglyoxal to S-D-lactoylglutathione, while glyoxalase II converts S-Dlactoylglutathione to D-lactate. Methylglyoxal is a toxic α-oxoaldehyde product formed by several metabolic pathways found in all cells. This study involves the preliminary characterization of a glyoxalase I protein from C. difficile through cloning of the gene from C. difficile 630 (CD3610), heterologous overexpression, purification, and analysis of the protein. This glyoxalase is of interest because expression has been shown to be increased in microarray data of C. difficile in response to different stresses. Five potential glyoxalase-related open reading frames (ORFs) were identified; CD3610 was unique in that the gene was upregulated in response to heat shock, acid shock, alkali shock and aerobic shock but had no change in response to antibiotics. In addition, this glyoxalase protein may be a nickel-containing metalloprotein. Nickel proteins are rare, and only eight have been described to date. Further understanding of the enzymology of this glyoxalase protein could potentially lead ii to a better understanding of the C. difficile organism. Biophysical characterization of this novel glyoxalase enzyme could lead to identification of a useful target for drug therapy. Preliminary characterization of the putative glyoxalase gene from C. difficile 630 involved cloning the CD3610 ORF insert into the expression vector pET24D. The plasmid containing the open reading frame was transformed into an expression strain of Escherichia coli, BL21(DE3). Preliminary purification of the putative glyoxalase protein was done through ion-exchange chromatography, glutathione affinity chromatography, and hydrophobic interaction chromatography. Biophysical characterization of the recombinant protein was done through SDS-PAGE to determine molecular weight. Functional characterization of the recombinant protein was determined using an in vitro glyoxalase I enzymatic assay, and metal activation studies. The CD3610 ORF was successfully cloned into a pET24d plasmid vector with five mutations. The mutation at position 2 was created because the restriction enzyme, NcoI was used to fuse the CD3610 gene in frame with the start codon. This mutation resulted in a conservative substitution, because isoleucine and valine are both hydrophobic amino acids. The CD3610 gene was overexpressed in a large scale volume of 3 L, in an E. coli expression strain, BL21(DE3). The experimental culture clearly overexpressed a huge amount of protein which correlated with the predicted molecular weight ~15 kDa. Unfortunately, the CD3610 protein was not completely purified and glyoxalase I enzymatic activity was not established.

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