Characterization of a putative trehalose biosynthetic pathway in salmonella typhimurium
- WCU Author/Contributor (non-WCU co-authors, if there are any, appear on document)
- Connor Joseph Larmore (Creator)
- Institution
- Western Carolina University (WCU )
- Web Site: http://library.wcu.edu/
- Advisor
- Amanda Storm
Abstract: Trehalose is a non-reducing disaccharide composed of two glucose monomers. In living organisms, this sugar is connected by an a-1,1-glycosidic linkage. In bacteria, trehalose is a component of various glycolipids and has been shown to confer abiotic stress tolerance. Genes believed to encode a trehalose biosynthetic pathway, including the enzymes maltooligosyl trehalose synthase (StMTSase) and maltooligosyl trehalose trehalohydrolase (StMTHase), have been identified in Salmonella typhimurium and shown by gene knockout to be essential for bacterial growth. However, the structure and activity of these types of enzymes have never been studied in any species of Proteobacteria. This study aimed to characterize the putative StMTSase and StMTHase enzymes. Comparative sequence and structure analysis revealed that both enzymes retain all the amino acids required for catalysis and are similar in overall architecture to functional homologs. Both enzymes also contain largely conserved substrate binding sites, although a tyrosine cluster identified in most other characterized MTSases is only partially conserved in the StMTSase. The putative StMTSase was biochemically characterized using the Nelson-Somogyi (NS) method for detecting reducing sugars, as MTSase enzymes convert reducing maltooligosaccharides into the non-reducing saccharide maltooligosyl trehalose. This enzyme was active on maltohexaose and dextrin but not maltotriose. It displayed the greatest activity at 40°C, a pH of 7.0, and in a citrate phosphate (McIlvaine) buffer containing potassium. Finally, two mutant StMTSases were produced using site-directed mutagenesis. One mutant contained substitutions of tyrosine at two positions (M460Y and V464Y) to match residues forming part of the tyrosine cluster that is conserved in most other characterized MTSases. This enzyme displayed no detectable activity. The other mutant contained a single substitution of tyrosine (M460Y) and displayed activity comparable to the wild-type enzyme. Overall, this study provides evidence that the putative MTSase/MTHase trehalose biosynthetic pathway identified in S. typhimurium is functional. This study contributes to a better understanding of two reportedly essential Salmonella genes and their products, which could potentially be developed into therapeutic targets.
Characterization of a putative trehalose biosynthetic pathway in salmonella typhimurium
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Created on 6/1/2023
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Additional Information
- Publication
- Thesis
- Language: English
- Date: 2023
- Subjects
- Biosynthesis
- Trehalose
- Salmonella typhimurium