The crystal structure of alanine racemase from Streptococcus pneumoniae, a target for structure-based drug design

<p>Abstract</p> <p>Background</p> <p><it>Streptococcus pneumoniae </it>is a globally important pathogen. The Gram-positive diplococcus is a leading cause of pneumonia, otitis media, bacteremia, and meningitis, and antibiotic resistant strains have become increasingly common over recent years.Alanine racemase is a ubiquitous enzyme among bacteria and provides the essential cell wall precursor, D-alanine. Since it is absent in humans, this enzyme is an attractive target for the development of drugs against <it>S. pneumoniae </it>and other bacterial pathogens.</p> <p>Results</p> <p>Here we report the crystal structure of alanine racemase from <it>S. pneumoniae </it>(Alr_SP). Crystals diffracted to a resolution of 2.0 Å and belong to the space group P3₁21 with the unit cell parameters a = b = 119.97 Å, c = 118.10 Å, α = β = 90° and γ = 120°. Structural comparisons show that Alr_SPshares both an overall fold and key active site residues with other bacterial alanine racemases. The active site cavity is similar to other Gram positive alanine racemases, featuring a restricted but conserved entryway.</p> <p>Conclusions</p> <p>We have solved the structure of Alr_SP, an essential step towards the development of an accurate pharmacophore model of the enzyme, and an important contribution towards our on-going alanine racemase structure-based drug design project. We have identified three regions on the enzyme that could be targeted for inhibitor design, the active site, the dimer interface, and the active site entryway.</p

Biochemical and structural characterization of alanine racemase from Bacillus anthracis (Ames)

<p>Abstract</p> <p>Background</p> <p><it>Bacillus anthracis </it>is the causative agent of anthrax and a potential bioterrorism threat. Here we report the biochemical and structural characterization of <it>B. anthracis </it>(Ames) alanine racemase (Alr_<it>Bax</it>), an essential enzyme in prokaryotes and a target for antimicrobial drug development. We also compare the native Alr_{<it>Bax </it>}structure to a recently reported structure of the same enzyme obtained through reductive lysine methylation.</p> <p>Results</p> <p><it>B. anthracis </it>has two open reading frames encoding for putative alanine racemases. We show that only one, <it>dal1</it>, is able to complement a D-alanine auxotrophic strain of <it>E. coli</it>. Purified Dal1, which we term Alr_<it>Bax</it>, is shown to be a dimer in solution by dynamic light scattering and has a V_maxfor racemization (L- to D-alanine) of 101 U/mg. The crystal structure of unmodified Alr_{<it>Bax </it>}is reported here to 1.95 Å resolution. Despite the overall similarity of the fold to other alanine racemases, Alr_{<it>Bax </it>}makes use of a chloride ion to position key active site residues for catalysis, a feature not yet observed for this enzyme in other species. Crystal contacts are more extensive in the methylated structure compared to the unmethylated structure.</p> <p>Conclusion</p> <p>The chloride ion in Alr_{<it>Bax </it>}is functioning effectively as a carbamylated lysine making it an integral and unique part of this structure. Despite differences in space group and crystal form, the two Alr_{<it>Bax </it>}structures are very similar, supporting the case that reductive methylation is a valid rescue strategy for proteins recalcitrant to crystallization, and does not, in this case, result in artifacts in the tertiary structure.</p

Purification and preliminary crystallization of alanine racemase from Streptococcus pneumoniae

<p>Abstract</p> <p>Background</p> <p>Over the past fifteen years, antibiotic resistance in the Gram-positive opportunistic human pathogen <it>Streptococcus pneumoniae </it>has significantly increased. Clinical isolates from patients with community-acquired pneumonia or otitis media often display resistance to two or more antibiotics. Given the need for new therapeutics, we intend to investigate enzymes of cell wall biosynthesis as novel drug targets. Alanine racemase, a ubiquitous enzyme among bacteria and absent in humans, provides the essential cell wall precursor, D-alanine, which forms part of the tetrapeptide crosslinking the peptidoglycan layer.</p> <p>Results</p> <p>The alanine racemases gene from <it>S. pneumoniae </it>(<it>alr</it>_<it>SP</it>) was amplified by PCR and cloned and expressed in <it>Escherichia coli</it>. The 367 amino acid, 39854 Da dimeric enzyme was purified to electrophoretic homogeneity and preliminary crystals were obtained. Racemic activity was demonstrated through complementation of an <it>alr </it>auxotroph of <it>E. coli </it>growing on L-alanine. In an alanine racemases photometric assay, specific activities of 87.0 and 84.8 U mg^-1were determined for the conversion of D- to L-alanine and L- to D-alanine, respectively.</p> <p>Conclusion</p> <p>We have isolated and characterized the alanine racemase gene from the opportunistic human pathogen <it>S. pneumoniae</it>. The enzyme shows sufficient homology with other alanine racemases to allow its integration into our ongoing structure-based drug design project.</p

Cryo-electron Microscopy Structure of the 70S Ribosome from Enterococcus faecalis

Enterococcus faecalis is a gram-positive organism responsible for serious infections in humans, but as with many bacterial pathogens, resistance has rendered a number of commonly used antibiotics ineffective. Here, we report the cryo-EM structure of the E. faecalis 70S ribosome to a global resolution of 2.8 Å. Structural differences are clustered in peripheral and solvent exposed regions when compared with Escherichia coli, whereas functional centres, including antibiotic binding sites, are similar to other bacterial ribosomes. Comparison of intersubunit conformations among five classes obtained after three-dimensional classification identifies several rotated states. Large ribosomal subunit protein bL31, which forms intersubunit bridges to the small ribosomal subunit, assumes different conformations in the five classes, revealing how contacts to the small subunit are maintained throughout intersubunit rotation. A tRNA observed in one of the five classes is positioned in a chimeric pe/E position in a rotated ribosomal state. The 70S ribosome structure of E. faecalisnow extends our knowledge of bacterial ribosome structures and may serve as a basis for the development of novel antibiotic compounds effective against this pathogen

Resolving the cofactor-binding site in the proline biosynthetic enzyme human pyrroline-5-carboxylate reductase 1

Pyrroline-5-carboxylate reductase (PYCR) is the final enzyme in proline biosynthesis, catalyzing the NAD(P)H-dependent reduction of [?]1-pyrroline-5-carboxylate (P5C) to proline. Mutations in the PYCR1 gene alter mitochondrial function and cause the connective tissue disorder cutis laxa. Furthermore, PYCR1 is overexpressed in multiple cancers, and the PYCR1 knock-out suppresses tumorigenic growth, suggesting that PYCR1 is a potential cancer target. However, inhibitor development has been stymied by limited mechanistic details for the enzyme, particularly in light of a previous crystallographic study that placed the cofactor-binding site in the C-terminal domain rather than the anticipated Rossmann fold of the N-terminal domain. To fill this gap, we report crystallographic, sedimentation- velocity, and kinetics data for human PYCR1. Structures of binary complexes of PYCR1 with NADPH or proline determined at 1.9 Å resolution provide insight into cofactor and substrate recognition.WeseeNADPHbound to the Rossmann fold, over 25 Å from the previously proposed site. The 1.85 Å resolution structure of a ternary complex containing NADPH and a P5C/proline analog provides a model of the Michaelis complex formed during hydride transfer. Sedimentation velocity shows that PYCR1 forms a concentration-dependent decamer in solution, consistent with the pentamer-of-dimers assembly seen crystallographically. Kinetic and mutational analysis confirmed several features seen in the crystal structure, including the importance of a hydrogen bond between Thr-238 and the substrate as well as limited cofactor discrimination

Gamma-Ray Emissions from Pulsars: Spectra of the TEV Fluxes from Outer-Gap Accelerators

We study the gamma-ray emissions from an outer-magnetospheric potential gap around a rotating neutron star. Migratory electrons and positrons are accelerated by the electric field in the gap to radiate copious gamma-rays via curvature process. Some of these gamma-rays materialize as pairs by colliding with the X-rays in the gap, leading to a pair production cascade. Imposing the closure condition that a single pair produces one pair in the gap on average, we explicitly solve the strength of the acceleration field and demonstrate how the peak energy and the luminosity of the curvature-radiated, GeV photons depend on the strength of the surface blackbody and the power-law emissions. Some predictions on the GeV emission from twelve rotation-powered pulsars are presented. We further demonstrate that the expected pulsed TeV fluxes are consistent with their observational upper limits. An implication of high-energy pulse phase width versus pulsar age, spin, and magnetic moment is discussed.Comment: Revised to compute absolute TeV spectra (22 pages, 9 figures

New Classes of Alanine Racemase Inhibitors Identified by High-Throughput Screening Show Antimicrobial Activity against Mycobacterium tuberculosis

In an effort to discover new drugs to treat tuberculosis (TB) we chose alanine racemase as the target of our drug discovery efforts. In Mycobacterium tuberculosis, the causative agent of TB, alanine racemase plays an essential role in cell wall synthesis as it racemizes L-alanine into D-alanine, a key building block in the biosynthesis of peptidoglycan. Good antimicrobial effects have been achieved by inhibition of this enzyme with suicide substrates, but the clinical utility of this class of inhibitors is limited due to their lack of target specificity and toxicity. Therefore, inhibitors that are not substrate analogs and that act through different mechanisms of enzyme inhibition are necessary for therapeutic development for this drug target.To obtain non-substrate alanine racemase inhibitors, we developed a high-throughput screening platform and screened 53,000 small molecule compounds for enzyme-specific inhibitors. We examined the 'hits' for structural novelty, antimicrobial activity against M. tuberculosis, general cellular cytotoxicity, and mechanism of enzyme inhibition. We identified seventeen novel non-substrate alanine racemase inhibitors that are structurally different than any currently known enzyme inhibitors. Seven of these are active against M. tuberculosis and minimally cytotoxic against mammalian cells.This study highlights the feasibility of obtaining novel alanine racemase inhibitor lead compounds by high-throughput screening for development of new anti-TB agents

Cryo‑electron microscopy structure of the 70S ribosome from Enterococcus faecalis

Enterococcus faecalis is a gram-positive organism responsible for serious infections in humans, but as with many bacterial pathogens, resistance has rendered a number of commonly used antibiotics ineffective. Here, we report the cryo-EM structure of the E. faecalis 70S ribosome to a global resolution of 2.8 Å. Structural differences are clustered in peripheral and solvent exposed regions when compared with Escherichia coli, whereas functional centres, including antibiotic binding sites, are similar to other bacterial ribosomes. Comparison of intersubunit conformations among five classes obtained after three-dimensional classification identifies several rotated states. Large ribosomal subunit protein bL31, which forms intersubunit bridges to the small ribosomal subunit, assumes different conformations in the five classes, revealing how contacts to the small subunit are maintained throughout intersubunit rotation. A tRNA observed in one of the five classes is positioned in a chimeric pe/E position in a rotated ribosomal state. The 70S ribosome structure of E. faecalis now extends our knowledge of bacterial ribosome structures and may serve as a basis for the development of novel antibiotic compounds effective against this pathogen

Massive migration from the steppe is a source for Indo-European languages in Europe

We generated genome-wide data from 69 Europeans who lived between 8,000-3,000 years ago by enriching ancient DNA libraries for a target set of almost four hundred thousand polymorphisms. Enrichment of these positions decreases the sequencing required for genome-wide ancient DNA analysis by a median of around 250-fold, allowing us to study an order of magnitude more individuals than previous studies and to obtain new insights about the past. We show that the populations of western and far eastern Europe followed opposite trajectories between 8,000-5,000 years ago. At the beginning of the Neolithic period in Europe, ~8,000-7,000 years ago, closely related groups of early farmers appeared in Germany, Hungary, and Spain, different from indigenous hunter-gatherers, whereas Russia was inhabited by a distinctive population of hunter-gatherers with high affinity to a ~24,000 year old Siberian6 . By ~6,000-5,000 years ago, a resurgence of hunter-gatherer ancestry had occurred throughout much of Europe, but in Russia, the Yamnaya steppe herders of this time were descended not only from the preceding eastern European hunter-gatherers, but from a population of Near Eastern ancestry. Western and Eastern Europe came into contact ~4,500 years ago, as the Late Neolithic Corded Ware people from Germany traced ~3/4 of their ancestry to the Yamnaya, documenting a massive migration into the heartland of Europe from its eastern periphery. This steppe ancestry persisted in all sampled central Europeans until at least ~3,000 years ago, and is ubiquitous in present-day Europeans. These results provide support for the theory of a steppe origin of at least some of the Indo-European languages of Europe

Role of Alanine Racemase Mutations in Mycobacterium tuberculosis d-Cycloserine Resistance.

A screening of more than 1,500 drug-resistant strains of Mycobacterium tuberculosis revealed evolutionary patterns characteristic of positive selection for three alanine racemase (Alr) mutations. We investigated these mutations using molecular modeling, in vitro MIC testing, as well as direct measurements of enzymatic activity, which demonstrated that these mutations likely confer resistance to d-cycloserine