Licentiate thesis, 2009
Enzymes in thymidylate synthesis in Ureaplasma parvum as medical targetsLin, Jay
AbstractThe wall less bacterium Ureaplasma parvum (Up) is associated with ureathritis in adults and pneumonia in neonates. Up lack de novo nucleotide synthesis genes and has to import all DNA precursors. This thesis investigates known DNA biosynthesis pathways as targets for new antibiotics and concerns two enzymes in Up thymidylate synthesis; a thymidylate synthase (TS) and thymidine kinase (UpTK). TS activity was detected in Up-extracts and UU572 DNA could rescue a TS mutant E. coli. UU572 appeared to be proteolytic cleaved and cell cycle regulated in Up. Codon modified UU572 was cloned for expression in E. coli. However, no protein expression could be detected. A codon optimized synthesized UU572 homolog; MPN358 from Mycoplasma pneumonia was expressed in E. coli and showed TS activity. Low sequence homology to existing TSs suggests that UU572 and its homologs, belong to a new class of TS enzymes, which may contribute to future antibiotic development in human and veterinary medicine. Thirteen click chemistry-synthesized 3´-triazole thymidine analogs (1-13), using AZT as backbone, were evaluated with UpTK and hTK1. The bacterial TK exhibited a more open 3D structure than hTK1 explaining its substrate efficiency, while hTK1 seemed to have more closed structure as reflected by higher inhibition by the analogs. Docking models with 13 in TK1 structures revealed amino acid substitutions in the active site and most likely explain the different enzyme specificity. In addition, molecular docking could explain the 6-fold higher inhibition by the nucleoside analog 3´-azido-methyl-deoxythymidine (AZMT) with UpTK compared to hTK1. Nucleoside analogs have been used for fighting viruses with minimal side-effects. Why not use this strategy to control bacterial infections? The results presented in this thesis contribute towards attaining this goal.
Keywordslic.-avh; enzymes; ureaplasma; biosynthesis; disease control; biochemistry
Publisher: Department of Anatomy, Physiology and Biochemistry, Swedish University of Agricultural Sciences
Swedish University of Agricultural Sciences, Department of Anatomy, Physiology and Biochemistry (AFB)
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