Investigating the structure, function & regulation of dihydrodipicolinate synthase from streptococcus pneumoniae

© 2011 Dr. Natalia Elena KetarenGiven the rise in drug-resistant Streptococcus pneumoniae, there is an urgent need to discover new antimicrobial agents targeting this human pathogen and an equally urgent need to characterise new drug targets. One such target is dihydrodipicolinate synthase (DHDPS), which catalyses the rate-limiting step in bacterial (S)-lysine biosynthesis. Hence, the overall aim of this dissertation is to characterise the structure, function and regulation of DHDPS from S. pneumoniae. This thesis commences with a thorough literature review in Chapter 1 describing the microbiology of S. pneumoniae, the incidence and mechanisms of antibiotic resistance and the importance and enzymology of (S)-lysine biosynthesis in bacteria. This includes an in-depth review of the structure, function and regulation of DHDPS from this important pathogen. Chapter 2 follows on by documenting a detailed account of the methodologies employed in the study to examine the overall aim of this dissertation. Chapter 3 provides a detailed review of the fundamental techniques used throughout the course of this study including circular dichroism (CD) spectroscopy, analytical ultracentrifugation (AUC), enzyme kinetics and X-ray crystallography. Chapter 4 describes the cloning of the DHDPS gene (dapA) from a clinical isolate of Streptococcus pneumoniae (OXC141 strain) and the strategy used to express, purify the recombinant enzyme. ~30 mg of recombinant protein was purified from 1 L of post- induced E. coli cultures, with a final specific activity of 1.61 U resulting from the 2-fold purification procedure. With the success of the recombinant protein production, solution studies, kinetic experiments and crystallization trials were performed in Chapter 5. The chapter begins firstly by generating a DHDPS gene knock out strain (ΔdapA) of Streptococcus pneumoniae that shows that ΔdapA is unable to grow unless supplemented with (S)- lysine. This result validates DHDPS as an antibiotic target in S. pneumoniae. The recombinant enzyme is active with a kcat = 150 s-1, KMPYR = 1.41 mM and KMASA = 0.13 mM and forms an α/β fold in solution similar to Escherichia coli DHDPS (Ec-DHDPS). However, thermal denaturation experiments monitored by circular dichroism spectroscopy demonstrate that Sp-DHDPS unfolds via a single transition with an apparent melting temperature (TM=app) of 71.7°C, which is significantly more thermostable than Ec-DHDPS (TMapp = 59.1°C). To further characterize the stability of the enzyme, concentration-dependent sedimentation equilibrium experiments were conducted in the analytical ultracentrifuge and show that Sp-DHDPS exists in a dimer- tetramer equilibrium with a KD 4→2 = 1.7 nM, which is considerably tighter than Ec- DHDPS (KD 4→2 = 76 nM). Upon characterization of the structural solution properties of Sp-DHDPS, Sp-DHDPS crystals were grown in high molecular weight PEG precipitants via the hanging-drop vapour diffusion method. The best crystal, from which data was collected, diffracted beyond 2.0 Å. The crystal structure of Sp-DHDPS was subsequently determined to 2.0 Å. The enzyme is shown to be tetrameric in the crystal state with dimensions of 105 " 62.4 " 106 Å, consistent with the biophysical measurements in solution. The active site contains almost identical composition and orientation of residues known to be important for catalytic activity in Ec-DHDPS. However, the tetramerisation interface of Sp-DHDPS is significantly different in composition and has greater buried surface area (790 Å2) compared to Ec-DHDPS (497 Å2). This explains why the Sp-DHDPS tetramer is considerably more thermally and thermodynamically stable than Ec-DHDPS. With the increase in drug-resistant bacteria being linked to the overuse and misuse of broad-spectrum antibiotics, there is a greater emphasis on targeting multiple sites on a potential drug target. Studies of the allosteric regulation of the enzyme are discussed in detail in Chapter 6. A key finding of this work is that Sp-DHDPS is the first Gram- positive DHDPS enzyme to demonstrate allosteric regulation by (S)-lysine. Through detailed kinetic characterisation, it was found that Sp-DHDPS exhibits high sensitivity to inhibition by (S)-lysine exceeding that of any other (S)-lysine-sensitive bacterial DHDPS characterised to date with KiPYR of 0.035 ± 0.002 mM and KiASA and Ki`ASA of 0.011 ± 0.011 mM and 0.026 ± 0.026 mM respectively. Thus, the degree of inhibition is similar to that found in plant DHDPS enzymes. To provide insight into the structural mechanism of the tight allosteric regulation of Sp-DHDPS, the crystal structure of Sp- DHDPS was determined, in complex with (S)-lysine. The (S)-lysine bound structure showed that significant conformational changes occur upon (S)-lysine binding. Similar to Ec-DHDPS and Nicotiana sylvestris DHDPS (Ns-DHDPS), two molecules of (S)- lysine were observed to bind in a bis-conformation in the allosteric cleft adjacent to the active site. However, unlike Ec-DHDPS and Ns-DHDPS, significant conformational changes at the tight-dimer interface assisted by the sharp rotation of H59 occur upon (S)- lysine binding. These changes resulted in the ‘contraction’, or ‘closing-in’ of the two monomers at the tight-dimer interface, leading to an increase in the number of contacts at this interface. In doing so, the presence of the two (S)-lysine molecules, with the addition of the contraction of the two monomers, serves to decrease the accessibility of the substrates to the active site, and/or attenuates the release of the products thus resulting is the inhibition of catalysis. In addition, a unique electrostatic interaction between the ligand and the enzyme is observed facilitated by the residue Glu62. The essentiality of the unique residues His59 and Glu62 were probed via mutagenesis, which revealed the alanine substitutions of these residues rendered the enzyme insensitive to (S)-lysine inhibition demonstrating a critical role in allostery for both residues. This study is the first to describe the knock-out phenotype, solution properties, and crystal structure of Sp-DHDPS. Chapter 7 summarizes the important findings of this dissertation that offers significant insight into future development of novel antimicrobial agents, targeting DHDPS from pathogenic bacteria such as S. pneumoniae