title: From knock-out phenotype to three-dimensional structure of a promising antibiotic target from Streptococcus pneumoniae.
abstract: Given the rise in drug-resistant Streptococcus pneumoniae, there is an urgent need to discover new antimicrobials targeting this pathogen and an equally urgent need to characterize new drug targets. A promising antibiotic target is dihydrodipicolinate synthase (DHDPS), which catalyzes the rate-limiting step in lysine biosynthesis. In this study, we firstly show by gene knock out studies that S. pneumoniae (sp) lacking the DHDPS gene is unable to grow unless supplemented with lysine-rich media. We subsequently set out to characterize the structure, function and stability of the enzyme drug target. Our studies show that sp-DHDPS is folded and active with a k(cat) = 22 s(-1), K(M)(PYR) = 2.55 ± 0.05 mM and K(M)(ASA) = 0.044 ± 0.003 mM. Thermal denaturation experiments demonstrate sp-DHDPS exhibits an apparent melting temperature (T(M)(app)) of 72 °C, which is significantly greater than Escherichia coli DHDPS (Ec-DHDPS) (T(M)(app) = 59 °C). Sedimentation studies show that sp-DHDPS exists in a dimer-tetramer equilibrium with a K(D)(4→2) = 1.7 nM, which is considerably tighter than its E. coli ortholog (K(D)(4→2) = 76 nM). To further characterize the structure of the enzyme and probe its enhanced stability, we solved the high resolution (1.9 Å) crystal structure of sp-DHDPS (PDB ID 3VFL). The enzyme is tetrameric in the crystal state, consistent with biophysical measurements in solution. Although the sp-DHDPS and Ec-DHDPS active sites are almost identical, the tetramerization interface of the s. pneumoniae enzyme is significantly different in composition and has greater buried surface area (800 Å(2)) compared to its E. coli counterpart (500 Å(2)). This larger interface area is consistent with our solution studies demonstrating that sp-DHDPS is considerably more thermally and thermodynamically stable than Ec-DHDPS. Our study describe for the first time the knock-out phenotype, solution properties, stability and crystal structure of DHDPS from S. pneumoniae, a promising antimicrobial target.
authors.affiliation: Department of Biochemistry, La Trobe Institute for Molecular Science, La Trobe University, Melbourne, Victoria, Australia ; Department of Biochemistry and Molecular Biology, Bio21 Molecular Science and Biotechnology Institute, University of Melbourne, Victoria, Australia.
authors.forename: Con
authors.initials: C
authors.lastname: Dogovski
authors.affiliation: St Vincent's Institute of Medical Research, Fitzroy, Victoria, Australia.
authors.forename: Michael A
authors.initials: MA
authors.lastname: Gorman
authors.affiliation: Department of Biochemistry and Molecular Biology, Bio21 Molecular Science and Biotechnology Institute, University of Melbourne, Victoria, Australia.
authors.forename: Natalia E
authors.initials: NE
authors.lastname: Ketaren
authors.affiliation: Department of Microbiology & Immunology, University of Melbourne, Victoria, Australia.
authors.forename: Judy
authors.initials: J
authors.lastname: Praszkier
authors.affiliation: Department of Biochemistry, La Trobe Institute for Molecular Science, La Trobe University, Melbourne, Victoria, Australia.
authors.forename: Leanne M
authors.initials: LM
authors.lastname: Zammit
authors.affiliation: Australian Synchrotron, Clayton, Victoria, Australia.
authors.forename: Haydyn D
authors.initials: HD
authors.lastname: Mertens
authors.affiliation: School of Applied Sciences, RMIT University, Melbourne, Victoria, Australia.
authors.forename: Gary
authors.initials: G
authors.lastname: Bryant
authors.affiliation: Department of Microbiology & Immunology, University of Melbourne, Victoria, Australia.
authors.forename: Ji
authors.initials: J
authors.lastname: Yang
authors.affiliation: Department of Biochemistry and Molecular Biology, Bio21 Molecular Science and Biotechnology Institute, University of Melbourne, Victoria, Australia.
authors.forename: Michael D W
authors.initials: MD
authors.lastname: Griffin
authors.affiliation: Biomolecular Interaction Centre and School of Biological Sciences, University of Canterbury, Christchurch, New Zealand.
authors.forename: F Grant
authors.initials: FG
authors.lastname: Pearce
authors.affiliation: Biomolecular Interaction Centre and School of Biological Sciences, University of Canterbury, Christchurch, New Zealand ; Callaghan Innovation, Lower Hutt, New Zealand.
authors.forename: Juliet A
authors.initials: JA
authors.lastname: Gerrard
authors.affiliation: Centre for Structural Biology, Institute of Fundamental Sciences, Massey University, Palmerston North, New Zealand.
authors.forename: Geoffrey B
authors.initials: GB
authors.lastname: Jameson
authors.affiliation: Department of Biochemistry and Molecular Biology, Bio21 Molecular Science and Biotechnology Institute, University of Melbourne, Victoria, Australia ; St Vincent's Institute of Medical Research, Fitzroy, Victoria, Australia.
authors.forename: Michael W
authors.initials: MW
authors.lastname: Parker
authors.affiliation: Department of Microbiology & Immunology, University of Melbourne, Victoria, Australia.
authors.forename: Roy M
authors.initials: RM
authors.lastname: Robins-Browne
authors.affiliation: Department of Biochemistry, La Trobe Institute for Molecular Science, La Trobe University, Melbourne, Victoria, Australia ; Department of Biochemistry and Molecular Biology, Bio21 Molecular Science and Biotechnology Institute, University of Melbourne, Victoria, Australia.
authors.forename: Matthew A
authors.initials: MA
authors.lastname: Perugini
mesh_terms: D000900:Anti-Bacterial Agents; D001426:Bacterial Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics; D018360:Crystallography, X-Ray; D016503:Drug Delivery Systems; D004926:Escherichia coli; D055785:Gene Knockdown Techniques; D006836:Hydro-Lyases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics; D020836:Protein Structure, Quaternary; D017434:Protein Structure, Tertiary; D013296:Streptococcus pneumoniae / Q000201:enzymology* / Q000235:genetics*
chemical_list: D000900:Anti-Bacterial Agents; D001426:Bacterial Proteins; D006836:Hydro-Lyases; C020040:4-hydroxy-tetrahydrodipicolinate synthase