Kurt L. Krause

The Alanine Racemase of Mycobacterium smegmatis Is Essential for Growth in the Absence of d-Alanine

Alanine racemase, encoded by the gene alr, is an important enzyme in the synthesis of d-alanine for peptidoglycan biosynthesis. Strains of Mycobacterium smegmatis with a deletion mutation of the alr gene were found to require d-alanine for growth in both rich and minimal media. This indicates that alanine racemase is the only source of d-alanine for cell wall biosynthesis in M. smegmatis and confirms alanine racemase as a viable target gene for antimycobacterial drug development.

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

Background 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. Methodology/Principal Findings 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. Conclusions/Significance This study highlights the feasibility of obtaining novel alanine racemase inhibitor lead compounds by high-throughput screening for development of new anti-TB agents.

Structural features and kinetic characterization of alanine racemase from Staphylococcus aureus (Mu50)

The tertiary structure and kinetic properties of alanine racemase from Staphylococcus aureus are described and compared to other related alanine racemase structures.

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

BackgroundBacillus anthracis is the causative agent of anthrax and a potential bioterrorism threat. Here we report the biochemical and structural characterization of B. anthracis (Ames) alanine racemase (AlrBax), an essential enzyme in prokaryotes and a target for antimicrobial drug development. We also compare the native AlrBaxstructure to a recently reported structure of the same enzyme obtained through reductive lysine methylation.ResultsB. anthracis has two open reading frames encoding for putative alanine racemases. We show that only one, dal1, is able to complement a D-alanine auxotrophic strain of E. coli. Purified Dal1, which we term AlrBax, is shown to be a dimer in solution by dynamic light scattering and has a Vmax for racemization (L- to D-alanine) of 101 U/mg. The crystal structure of unmodified AlrBaxis reported here to 1.95 Å resolution. Despite the overall similarity of the fold to other alanine racemases, AlrBaxmakes 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.ConclusionThe chloride ion in AlrBaxis 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 AlrBaxstructures 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.

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

BackgroundStreptococcus pneumoniae 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 S. pneumoniae and other bacterial pathogens.ResultsHere we report the crystal structure of alanine racemase from S. pneumoniae (AlrSP). 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 AlrSP shares 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.ConclusionsWe have solved the structure of AlrSP, 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.

Investigation of the essentiality of glutamate racemase in Mycobacterium smegmatis.

The mycobacterial cell wall frequently has been used as a target for drug development, and d-glutamate, synthesized by glutamate racemase (MurI), is an important component of peptidoglycan. While the essentiality of the murI gene has been shown in several bacterial species, including Escherichia coli, Bacillus anthracis, and Streptococcus pneumoniae, studies in mycobacteria have not yet provided definitive results. This study aimed to determine whether murI is indeed essential and can serve as a possible target for structure-aided drug design. We have achieved this goal by creating a ΔmurI strain of Mycobacterium smegmatis, a close relative of Mycobacterium tuberculosis. The deletion of the murI gene in M. smegmatis could be achieved only in minimal medium supplemented with D-glutamate, demonstrating that MurI is essential for growth and that glutamate racemase is the only source of D-glutamate for peptidoglycan synthesis in M. smegmatis.

Structural plasticity and in vivo activity of Cas1 from the type I-F CRISPR–Cas system

CRISPR-Cas systems are adaptive immune systems in prokaryotes that provide protection against viruses and other foreign DNA. In the adaptation stage, foreign DNA is integrated into CRISPR (clustered regularly interspaced short palindromic repeat) arrays as new spacers. These spacers are used in the interference stage to guide effector CRISPR associated (Cas) protein(s) to target complementary foreign invading DNA. Cas1 is the integrase enzyme that is central to the catalysis of spacer integration. There are many diverse types of CRISPR-Cas systems, including type I-F systems, which are typified by a unique Cas1-Cas2-3 adaptation complex. In the present study we characterize the Cas1 protein of the potato phytopathogen Pectobacterium atrosepticum, an important model organism for understanding spacer acquisition in type I-F CRISPR-Cas systems. We demonstrate by mutagenesis that Cas1 is essential for adaptation in vivo and requires a conserved aspartic acid residue. By X-ray crystallography, we show that although P. atrosepticum Cas1 adopts a fold conserved among other Cas1 proteins, it possesses remarkable asymmetry as a result of structural plasticity. In particular, we resolve for the first time a flexible, asymmetric loop that may be unique to type I-F Cas1 proteins, and we discuss the implications of these structural features for DNA binding and enzymatic activity.

The structure of alanine racemase from Acinetobacter baumannii.

The crystal structure of A. baumannii alanine racemase (AlrAba) from the highly antibiotic resistant NCTC13302 strain has been solved to 1.9 Å resolution. Comparison of (AlrAba) with alanine racemases from closely related bacteria demonstrates a conserved overall fold.

Overexpression of a newly identified d-amino acid transaminase in Mycobacterium smegmatis complements glutamate racemase deletion

Glutamate racemase (MurI) has been proposed as a target for anti‐tuberculosis drug development based on the inability of ΔmurI mutants of Mycobacterium smegmatis to grow in the absence of d‐glutamate. In this communication, we identify ΔmurI suppressor mutants that are detected during prolonged incubation. Whole genome sequencing of these ΔmurI suppressor mutants identified the presence of a SNP, located in the promoter region of MSMEG_5795. RT‐qPCR and transcriptional fusion analyses revealed that the ΔmurI suppressor mutant overexpressed MSMEG_5795 14‐fold compared to the isogenic wild‐type. MSMEG_5795, which is annotated as 4‐amino‐4‐deoxychorismate lyase (ADCL) but which also has homology to d‐amino acid transaminase (d‐AAT), was expressed, purified and found to have d‐AAT activity and to be capable of producing d‐glutamate from d‐alanine. Consistent with its d‐amino acid transaminase function, overexpressed MSMEG_5795 is able to complement both ΔmurI deletion mutants and alanine racemase (Δalr) deletion mutants, thus confirming a multifunctional role for this enzyme in M. smegmatis.

Crystallization and preliminary X-ray analysis of the chemokine-binding protein from orf virus (Poxviridae)

The parapoxvirus orf virus (ORFV) encodes a chemokine-binding protein (CBP) that functions to downregulate the hosts immune response at the site of infection by blocking the chemokine-induced recruitment of immune cells. In order to shed light on the structural determinants of CBP-chemokine binding, ORFV CBP was crystallized as part of an ongoing structure-function study on this protein. ORFV CBP crystals were obtained by the sitting-drop vapour-diffusion technique using ammonium citrate as a precipitant. The crystal quality was greatly improved through the addition of small-molecule additives to the crystallization mother liquor. ORFV CBP crystals diffracted X-rays to 2.50 A resolution and belonged to the hexagonal space group P6(1)22 or its enantiomorph P6(5)22, with unit-cell parameters a = b = 75.62, c = 282.49 A, alpha = 90, beta = 90, gamma = 120 degrees.