From knock-out phenotype to three-dimensional structure of a promising antibiotic target from Streptococcus pneumoniae.

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.

Control of replication in I-complex plasmids.

The closely related plasmids that make up the I-complex group and the more distantly related IncL/M plasmids regulate the frequency of initiation of their replication by controlling the efficiency of translation of the rate limiting replication initiator protein, RepA. Translation initiation of repA is dependent on the formation of a pseudoknot immediately upstream of its Shine-Dalgarno sequence. Formation of this pseudoknot involves base pairing between two complementary sequences in the repA mRNA and requires that the secondary structure sequestering the distal sequence be disrupted by movement of the ribosome translating and terminating a leader peptide, whose coding sequence precedes and overlaps that of repA. Expression of repA is controlled by a small antisense RNA, RNAI, which on binding to its complementary target in the repA mRNA not only pre-empts formation of the pseudoknot, but also inhibits translation of the leader peptide. The requirement that translation of the leader peptide be completed for the pseudoknot to form increases the time available for the inhibitory interaction of RNAI with its target, so that at high copy number the frequency of pseudoknot formation is lowered, reducing the proportion of repA mRNA that are translated. At low copy number, when concentration of RNAI is low, repA is translated with increased frequency, leading to increased frequency of plasmid replication.