title: Comparative structure and function analyses of native and his-tagged forms of dihydrodipicolinate reductase from methicillin-resistant Staphylococcus aureus.
abstract: Given the rise of multi drug resistant bacterial strains, such as methicillin-resistant Staphylococcus aureus (MRSA), there is an urgent need to discover new antimicrobial agents. A validated but as yet unexplored target for new antibiotics is dihydrodipicolinate reductase (DHDPR), an enzyme that catalyzes the second step of the lysine biosynthesis pathway in bacteria. We report here the cloning, expression and purification of N-terminally his-tagged recombinant DHDPR from MRSA (6H-MRSA-DHDPR) and compare its secondary and quaternary structure with the wild type (MRSA-DHDPR) enzyme. Comparative analyses demonstrate that recombinant 6H-MRSA-DHDPR is folded and adopts the native tetrameric quaternary structure in solution. Furthermore, kinetic studies show 6H-MRSA-DHDPR is functional, displaying parameters for K(m)(NADH) of 6.0 μM, K(m)(DHDP) of 22 μM, and k(cat) of 21s(-1), which are similar to those reported for the native enzyme. The solution properties and stability of the 6H-MRSA-DHDPR enzyme are also reported in varying physicochemical conditions.
authors: Department of Biochemistry, La Trobe Institute for Molecular Science, La Trobe University, Melbourne, Victoria 3086, Australia.
authors: Con
authors: C
authors: Dogovski
authors: Sudhir R
authors: SR
authors: Dommaraju
authors: Liam C
authors: LC
authors: Small
authors: Matthew A
authors: MA
authors: Perugini
mesh_terms: D003001:Cloning, Molecular; D050746:Dihydrodipicolinate Reductase / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D004795:Enzyme Stability; D006639:Histidine / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D007700:Kinetics; D055624:Methicillin-Resistant Staphylococcus aureus / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics; D009994:Osmolar Concentration; D011487:Protein Conformation; D017510:Protein Folding; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism
chemical_list: D011994:Recombinant Proteins; D006639:Histidine; D050746:Dihydrodipicolinate Reductase