title: Catalytic mechanism and cofactor preference of dihydrodipicolinate reductase from methicillin-resistant Staphylococcus aureus.
abstract: Given the rapid rise in antibiotic resistance, including methicillin resistance in Staphylococcus aureus (MRSA), there is an urgent need to characterize novel drug targets. Enzymes of the lysine biosynthesis pathway in bacteria are examples of such targets, including dihydrodipicolinate reductase (DHDPR, E.C. 1.3.1.26), which is the product of an essential bacterial gene. DHDPR catalyzes the NAD(P)H-dependent reduction of dihydrodipicolinate (DHDP) to tetrahydrodipicolinate (THDP) in the lysine biosynthesis pathway. We show that MRSA-DHDPR exhibits a unique nucleotide specificity utilizing NADPH (K(m)=12μM) as a cofactor more effectively than NADH (K(m)=26μM). However, the enzyme is inhibited by high concentrations of DHDP when using NADPH as a cofactor, but not with NADH. Isothermal titration calorimetry (ITC) studies reveal that MRSA-DHDPR has ∼20-fold greater binding affinity for NADPH (K(d)=1.5μM) relative to NADH (K(d)=29μM). Kinetic investigations in tandem with ITC studies show that the enzyme follows a compulsory-order ternary complex mechanism; with inhibition by DHDP through the formation of a nonproductive ternary complex with NADP(+). This work describes, for the first time, the catalytic mechanism and cofactor preference of MRSA-DHDPR, and provides insight into rational approaches to inhibiting this valid antimicrobial target.
authors.affiliation: Department of Biochemistry and Molecular Biology, Bio21 Molecular Science and Biotechnology Institute, The University of Melbourne, Parkville, Victoria, Australia.
authors.forename: Sudhir R
authors.initials: SR
authors.lastname: Dommaraju
authors.forename: Con
authors.initials: C
authors.lastname: Dogovski
authors.forename: Peter E
authors.initials: PE
authors.lastname: Czabotar
authors.forename: Lilian
authors.initials: L
authors.lastname: Hor
authors.forename: Brian J
authors.initials: BJ
authors.lastname: Smith
authors.forename: Matthew A
authors.initials: MA
authors.lastname: Perugini
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D002151:Calorimetry; D002384:Catalysis; D050746:Dihydrodipicolinate Reductase / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D007700:Kinetics; D055624:Methicillin-Resistant Staphylococcus aureus / Q000201:enzymology* / Q000235:genetics; D008969:Molecular Sequence Data; D009243:NAD / Q000378:metabolism; D009249:NADP / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D013816:Thermodynamics
chemical_list: D001426:Bacterial Proteins; D009243:NAD; D009249:NADP; D050746:Dihydrodipicolinate Reductase