List of PhD student oral presentations

s oral PhD presentations PhD 1 The impact of residues 119 and 228 in the Tripoli metallo-β-lactamase TMB-1 involved in resistance to β-lactam antibiotics Susann Skagseth, Ørjan Samuelsen, Hanna-Kirsti S. Leiros 1 The Norwegian Structural Biology Centre (Norstruct), Department of Chemistry, UiT – The Arctic University of Norway, Tromsø, Norway 2 Department of Pharmacy, UiT – The Arctic University of Norway, Tromsø, Norway 3 Norwegian National Advisory Unit on Detection of Antimicrobial Resistance, University Hospital of North Norway, Tromsø, Norway Metallo-β-lactamases (MBLs) are enzymes with the ability to hydrolyze β-lactam antibiotics including penicillins, cephalosporins, and carbapenems resulting in bacterial strains resistant to virtually all βlactams. The worldwide dissemination of these MBLs in Gram-negative bacteria poses an increasing clinical threat. The MBL TMB-1 (Tripoli Metallo-β-lactamase) was initially discovered in a Achromobacter xylosoxidans isolate [1]. Subsequently, TMB-2 from Acinetobacter spp. was detected differing from TMB-1 with one single mutation (S228P). Residue E119 is close to the active site, and inbetween a conserved H116xHxD120 motif. Mutation at position 228 has shown to affect the catalytic efficiency in GIM-1 MBL [2], while mutation at 119 has only been studied in NDM-1 MBL [3]. In this study, three site-directed mutations on TMB-1 were made; E119Q/S/A, and TMB-1, mutants and TMB-2 from synthetically made genes. The TMB-1 and TMB-1 varients were expressed and purified using affinity and ion exchange purification. Thermofluor stability measurements and enzyme kinetic studies have been performed on TMB-1 and the TMB-1 variants. Crystals were obtained of TMB-1. The thermal stability measurements indicated that TMB-1 and variants stabilizes with 1 M NaCl. The TMB-1 mutants show in general a reduced catalytic efficiency compared to the TMB-1. The results showed that the substitution of S228 and E119 had an impact on the catalytic properties of the enzyme.