The Human Pathogen Clostridium Difficile: A Look at a Putative Glyoxalase Involved in Protection from Oxidative Stress
Date of Award
Master of Science in Biomedical Sciences
Francis E. Jenney, Jr PhD
Harry Kominsky, PhD
Kimberly Deadmond, Phd
Background: Hospital acquired infections due to Clostridium dijficile (C. diff) are associated with nosocomial diarrhea and pseudomembranous colitis. Using a published microarray analysis of C. diffstrain 630, several open reading frames (ORFs) were noted for their upregulation under different enviromnental stresses. One of these genes (CD1134) is a putative glyoxalase I. The glyoxalase enzymes detoxify a side product of glycolysis, methylglyoxal, and use metals as a cofactor.
Objectives: To express, purify and characterize CD1134, a putative glyoxalase I protein from Clostridium dijficile 630.
Methods: Using bioinformatic analysis we compared known glyoxalase genes from other species with the putative glyoxalase I of C. diff. The ORF CD 1134 was cloned into an expression vector and the protein was overexpressed in Escherichia coli and purified. Mass spectrometry (MS) was used to determine the molecular weight of the as-purified protein. Inductively Coupled Plasma MS was used to determine the metals bound to the as-purified protein. A metal activation study was conducted with several metal chlorides to observe the effect each metal has on enzyme activity. The recombinant enzyme was further characterized for optimal pH and substrate specificity.
Results: Through bioinformatic analysis it was observed that putative glyoxalase I CD 1134 shared the same conserved metal binding residues as those found in Glxi of Escherichia coli and Pseudomonas aeruginosa. Based on other studies of glyoxalase I both E. coli and P. aeruginosa proteins are nickel activated enzymes. We found that the ORF CD1134 of C. difficile 630 does appear to possess glyoxalase activity in vitro and appears also to be a nickel activated enzyme with an optimal pH at 6.5. ix
Lee, Tashinea, "The Human Pathogen Clostridium Difficile: A Look at a Putative Glyoxalase Involved in Protection from Oxidative Stress" (2012). PCOM Biomedical Studies Student Scholarship. 24.