Characterization of the diverse substrate specificities and biological roles of polyamine biosynthetic

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Title: Characterization of the diverse substrate specificities and biological roles of polyamine biosynthetic
Author: Lee, Jeongmi
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 .
URI: http : / /hdl .handle .net /2152 .5 /327
Date: 2008-09-18


Characterization of the diverse substrate specificities and biological roles of polyamine biosynthetic. Graduate School of Biomedical Sciences. Available electronically from http : / /hdl .handle .net /2152 .5 /327 .

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