| Title | From knock-out phenotype to three-dimensional structure of a promising antibiotic target from Streptococcus pneumoniae |
| Publication Type | Journal Article |
| Year of Publication | 2013 |
| Authors | Dogovski, C., Gorman M.A., Ketaren N.E., Praszkier J., Zammit L.M., Mertens H.D., Bryant G., Yang J., Griffin M.D.W., Pearce F.G., Gerrard J.A., Jameson G.B., Parker M.W., Robins-Browne R.M., and Perugini M.A. |
| Journal | PLoS ONE |
| Volume | 8 |
| Issue | 12 |
| Date Published | 2013 |
| ISSN | 19326203 (ISSN) |
| Keywords | Anti-Bacterial Agents, article, Bacterial Proteins, cell density, cell size, circular dichroism, controlled study, crystal structure, Crystallography, X-Ray, dapA gene, DHDPS gene, dihydrodipicolinate synthase, dipicolinic acid, Drug Delivery Systems, drug targeting, enzyme active site, enzyme conformation, enzyme denaturation, enzyme mechanism, Enzyme Stability, Escherichia coli, gene, gene inactivation, Gene Knockdown Techniques, Hydro-Lyases, hydrodynamics, nonhuman, protein function, protein secondary structure, Protein Structure, Quaternary, Protein Structure, Tertiary, Streptococcus pneumoniae, structure activity relation, synthetase, thermostability, unclassified drug |
| 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 kcat = 22 s-1, KM 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 (TM app) of 72°C, which is significantly greater than Escherichia coli DHDPS (Ec-DHDPS) (TMapp = 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 (KD 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 Å2) compared to its E. coli counterpart (500 Å2). 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. © 2013 Dogovski et al. |
| URL | http://www.scopus.com/inward/record.url?eid=2-s2.0-84892534088&partnerID=40&md5=d0ce196d3ae702f940b07576c678fa92 |
| DOI | 10.1371/journal.pone.0083419 |
