My research focuses on several enzymes expressed by the Gram positive bacteria Enterococci. These enzymes include the flavoproteins NADH peroxidase, NADH oxidase and -glycerophosphate oxidase. These have all been cloned, sequenced and overexpressed in E. coli. The enzymic mechanisms of these three enzymes are being studied by a combination of traditional spectrophotometric measurements and the creation of site-directed mutants to dissect the enzymatic reactions. The genetic regulation of glycerol metabolism in Enterococcus is also being studied. This involves the investigation of transcriptional control of the glp operon, which has been cloned. This study includes measuring transcription (mRNA) levels and the use of reporter genes fused to the glp operon. promoter. Another way in which glycerol metabolism is controlled is by phosphorylation of glycerol kinase, which activates the kinase and hence controls the flux through the catabolic pathway. The gene for enterococcal glycerol kinase has been closed and overexpressed in E. coli. This protein is now being used to study the control of enzyme activity within the cell.
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