Characterization of the Diverse Substrate Specificities and Biological Roles of Polyamine Biosynthetic Enzymes in Microorganisms
| dc.contributor.advisor | Phillips, Margaret A. | en |
| dc.creator | Lee, Jeongmi | en |
| dc.date.accessioned | 2010-07-12T17:36:07Z | |
| dc.date.available | 2010-07-12T17:36:07Z | |
| dc.date.issued | 2008-09-18 | |
| dc.description.abstract | The beta /alpha -barrel fold-type basic amino acid decarboxylases include eukaryotic ornithine decarboxylases (ODC), and bacterial and plant enzymes with activity on L-arginine and meso-diaminopimelate. These enzymes catalyze essential steps in polyamine and lysine biosynthesis. Phylogenetic analysis suggests that diverse bacterial species also contain ODC-like enzymes from this fold-type. However, in comparison to the eukaryotic ODCs, amino acid differences were identified in the sequence of the 310-helix that forms a key specificity element in the active site, suggesting they might function on novel substrates. Putative decarboxylases from a phylogenetically diverse range of bacteria were characterized to determine their substrate preference. Enzymes from species within Methanosarcina, Pseudomonas, Bartonella, Nitrosomonas, Thermotoga and Aquifex showed a strong preference for L-ornithine, while the enzyme from Vibrio vulnificus (VvL/ODC) had dual specificity functioning well on both L-ornithine and L-lysine. The X-ray structure of VvL/ODC was solved in the presence of the reaction products putrescine and cadaverine to 1.7 and 2.15 Å, respectively. The overall structure is similar to eukaryotic ODC, however reorientation of the 310-helix enlarges the substrate binding-pocket, thereby allowing L-lysine to be accommodated. The structure of the putrescine-bound enzyme suggests that a bridging water molecule between the shorter L-ornithine and key active site residues provides the structural basis for VvL/ODC to also function on this substrate. Our data demonstrate that there is greater structural and functional diversity in bacterial polyamine biosynthetic decarboxylases than previously suspected. The beta /alpha -barrel fold decarboxylases also include a group of bacterial putative carboxynorspermidine decarboxylases (CANSDCs), which were hypothesized to be involved in norspermidine biosynthesis. Putative norspermidine biosynthetic enzymes including V.vulnificus CANSDC were characterized to determine their substrate specificities. The polyamine biosynthetic pathway in V.cholerae was confirmed by in vivo reconstitution of norspermidine biosynthetic genes in E.coli. Knockout of polyamine biosynthetic genes in V.cholerae revealed that normal levels of norspermidine are not essential for cell viability and that norspermidine is important for biofilm formation as a signaling molecule in more than one regulatory pathways for this process. Our preliminary data from real time RT-PCR suggest that norspermidine may also be related to quorum sensing and virulence gene expressions in V.cholerae . | en |
| dc.format.digitalOrigin | born digital | en |
| dc.format.medium | Electronic | en |
| dc.format.mimetype | application/pdf | en |
| dc.identifier.oclc | 302285157 | |
| dc.identifier.uri | https://hdl.handle.net/2152.5/327 | |
| dc.language.iso | en | en |
| dc.subject | Lysine | en |
| dc.subject | Biosynthesis, Peptide | en |
| dc.subject | Polyamines | en |
| dc.title | Characterization of the Diverse Substrate Specificities and Biological Roles of Polyamine Biosynthetic Enzymes in Microorganisms | en |
| dc.type | Thesis | en |
| dc.type.genre | dissertation | en |
| dc.type.material | Text | en |
| thesis.date.available | 2009-09-18 | |
| thesis.degree.department | Graduate School of Biomedical Sciences | en |
| thesis.degree.discipline | Molecular Biophysics | en |
| thesis.degree.grantor | UT Southwestern Medical Center | en |
| thesis.degree.level | Doctoral | en |
| thesis.degree.name | Doctor of Philosophy | en |