One in a million: flow cytometric sorting of single cell-lysate assays in monodisperse picolitre double emulsion droplets for directed evolution.

Directed evolution relies on iterative cycles of randomization and selection. The outcome of an artificial evolution experiment is crucially dependent on (i) the numbers of variants that can be screened and (ii) the quality of the assessment of each clone that forms the basis for selection. Compartmentalization of screening assays in water-in-oil emulsion droplets provides an opportunity to screen vast numbers of individual assays with good signal quality.

A single mutation in the core domain of the lac repressor reduces leakiness.

Background: The lac operon provides cells with the ability to switch from glucose to lactose metabolism precisely when necessary. This metabolic switch is mediated by the lac repressor (LacI), which in the absence of lactose binds to the operator DNA sequence to inhibit transcription. Allosteric rearrangements triggered by binding of the lactose isomer allolactose to the core domain of the repressor impede DNA binding and lift repression.

A fully unsupervised compartment-on-demand platform for precise nanoliter assays of time-dependent steady-state enzyme kinetics and inhibition.

The ability to miniaturize biochemical assays in water-in-oil emulsion droplets allows a massive scale-down of reaction volumes, so that high-throughput experimentation can be performed more economically and more efficiently. Generating such droplets in compartment-on-demand (COD) platforms is the basis for rapid, automated screening of chemical and biological libraries with minimal volume consumption. Herein, we describe the implementation of such a COD platform to perform high precision nanoliter assays.

Droplets as reaction compartments for protein nanotechnology.

Extreme miniaturization of biological and chemical reactions in pico- to nanoliter microdroplets is emerging as an experimental paradigm that enables more experiments to be carried out with much lower sample consumption, paving the way for high-throughput experiments. This review provides the protein scientist with an experimental framework for (a) formation of polydisperse droplets by emulsification or, alternatively, of monodisperse droplets using microfluidic devices; (b) construction of experimental rigs and microfluidic chips for this purpose; and (c) handling and analysis of droplets.

A simple method to evaluate the biochemical compatibility of oil/surfactant mixtures for experiments in microdroplets.

The enormous reduction of assay volume afforded by compartmentalization into picolitre water-in-oil droplets is an exciting prospect for high-throughput biology. Maintaining the activity of encapsulated proteins is critical for experimental success, for example in in vitro directed evolution, where protein variants are expressed in droplets to identify mutants with improved properties. Here, we present a simple and rapid method to quantitatively compare concentrations of fluorescent molecules in microdroplets.

A tetrameric structure is not essential for activity in dihydrodipicolinate synthase (DHDPS) from Mycobacterium tuberculosis.

Dihydrodipicolinate synthase (DHDPS) is a validated antibiotic target for which a new approach to inhibitor design has been proposed: disrupting native tetramer formation by targeting the dimer-dimer interface. In this study, rational design afforded a variant of Mycobacterium tuberculosis, Mtb-DHDPS-A204R, with disrupted quaternary structure. X-ray crystallography (at a resolution of 2.

LC-MS and NMR characterization of the purple chromophore formed in the o-aminobenzaldehyde assay of dihydrodipicolinate synthase.

The enzyme dihydrodipicolinate synthase (DHDPS) has been widely investigated as a target for new antibiotics. The o-aminobenzaldehyde (o-ABA) assay is routinely used as a highly specific, if qualitative, tool for DHDPS purification, whereby fractions containing active DHDPS appear purple upon addition of o-ABA. The purple adduct absorbs in the visible region (540 nm) but has never been characterized in the 50 years since it was first reported.

Microfluidic droplets: new integrated workflows for biological experiments.

Miniaturization of the classical test tube to picoliter dimensions is possible in monodisperse water-in-oil droplets that are generated in microfluidic devices. The establishment of standard unit operations for droplet handling and the ability to carry out experiments with DNA, proteins, cells and organisms provides the basis for the design of more complex workflows to address biological challenges. The emerging experimental format makes possible a quantitative readout for large numbers of experiments with a precision comparable to the macroscopic scale.

NMR studies uncover alternate substrates for dihydrodipicolinate synthase and suggest that dihydrodipicolinate reductase is also a dehydratase.

Despite extensive effort, the drug target dihydrodipicolinate synthase (DHDPS) continues to evade effective inhibition. We used NMR spectroscopy to examine the substrate specificity of this enzyme and found that two pyruvate analogues previously classified as weak competitive inhibitors were turned over productively by DHDPS. Four other analogues were confirmed not to be substrates.

How essential is the 'essential' active-site lysine in dihydrodipicolinate synthase?

Dihydrodipicolinate synthase (DHDPS, E.C. 4.

The C-terminal regulatory domain is required for catalysis by Neisseria meningitidis alpha-isopropylmalate synthase.

alpha-Isopropylmalate synthase (alpha-IPMS) catalyses the first committed step in leucine biosynthesis in many pathogenic bacteria, including Neisseria meningitidis. This enzyme (NmeIPMS) has been purified, characterised, and compared to alpha-IPMS proteins from other bacteria. NmeIPMS is a homodimer which catalyses the condensation of alpha-ketoisovalerate (alpha-KIV) and acetyl coenzyme A (AcCoA), and is inhibited by leucine.

The quaternary structure of Escherichia coli N-acetylneuraminate lyase is essential for functional expression.

As part of a general study into the impact of quaternary structure on enzyme function, a library of 31 point mutations were engineered at the dimer-dimer interface of the homotetrameric (beta/alpha)(8)-barrel protein, N-acetylneuraminate lyase (NAL, EC 4.1.3.

The C-terminal domain of Escherichia coli dihydrodipicolinate synthase (DHDPS) is essential for maintenance of quaternary structure and efficient catalysis.

Dihydrodipicolinate synthase (DHDPS) catalyses the first committed step in the biosynthesis of (S)-lysine, an essential constituent of bacterial cell walls. Escherichia coli DHDPS is homotetrameric, and each monomer contains an N-terminal (alpha/beta)(8)-barrel, responsible for catalysis and regulation, and three C-terminal alpha-helices, the function of which is unknown. This study investigated the C-terminal domain of E.

Cloning and characterisation of dihydrodipicolinate synthase from the pathogen Neisseria meningitidis.

Neisseria meningitidis is an obligate commensal bacterium of humans, and also an important human pathogen. To facilitate future drug studies, we report here the biochemical and structural characterisation of a key enzyme in (S)-lysine biosynthesis, dihydrodipicolinate synthase (DHDPS), from N. meningitidis (NmeDHDPS).

The role of quaternary structure in (beta/alpha)(8)-barrel proteins: evolutionary happenstance or a higher level of structure-function relationships?

Despite significant effort, the role played by quaternary structure in enzymes often remains poorly understood. The (beta/alpha)(8)-barrel fold is a versatile scaffold that has been employed in a wide variety of organisms to catalyse a broad spectrum of reactions, and thus is a convenient motif for further investigation into the importance and role of quaternary structure. This review will highlight studies in which the interplay between catalytic activity and quaternary structure in the (beta/alpha)(8)-barrel family have been explored, revealing examples in which homo-oligomeric structure completes the active site or apparently enhances thermal stability.

The high-resolution structure of dihydrodipicolinate synthase from Escherichia coli bound to its first substrate, pyruvate.

Dihydrodipicolinate synthase (DHDPS) mediates the key first reaction common to the biosynthesis of (S)-lysine and meso-diaminopimelate, molecules which play a crucial cross-linking role in bacterial cell walls. An effective inhibitor of DHDPS would represent a useful antibacterial agent; despite extensive effort, a suitable inhibitor has yet to be found. In an attempt to examine the specificity of the active site of DHDPS, the enzyme was cocrystallized with the substrate analogue oxaloacetate.

Synthetic and biological studies on the spiro-mamakone system.

An exploration of the chemistry of the spiro-mamakone system, exemplified by the cytotoxic, fungal metabolite spiro-mamakone A, is presented. The first reported synthesis of the spiro-mamakone carbon skeleton was achieved, as well as the synthesis of a variety of closely related analogues of the natural product. Biological testing of the synthetic analogues generated a structure-activity profile for the natural product, establishing the importance of the enedione moiety to biological activity.

Conserved main-chain peptide distortions: a proposed role for Ile203 in catalysis by dihydrodipicolinate synthase.

In recent years, dihydrodipicolinate synthase (DHDPS, E.C. 4.

Crystal structure and kinetic study of dihydrodipicolinate synthase from Mycobacterium tuberculosis.

The three-dimensional structure of the enzyme dihydrodipicolinate synthase (KEGG entry Rv2753c, EC 4.2.1.

Inhibiting dihydrodipicolinate synthase across species: towards specificity for pathogens?

Dihydrodipicolinate synthase (DHDPS) is a key enzyme in lysine biosynthesis and an important antibiotic target. The specificity of a range of heterocyclic product analogues against DHDPS from three pathogenic species, Bacillus anthracis, Mycobacterium tuberculosis and methicillin-resistant Staphylococcus aureus, and the evolutionarily related N-acetylneuraminate lyase, has been determined. The results suggest that the development of species-specific inhibitors of DHDPS as potential antibacterials is achievable.

Role of arginine 138 in the catalysis and regulation of Escherichia coli dihydrodipicolinate synthase.

In plants and bacteria, the branch point of (S)-lysine biosynthesis is the condensation of (S)-aspartate-beta-semialdehyde [(S)-ASA] and pyruvate, a reaction catalyzed by dihydrodipicolinate synthase (DHDPS, EC 4.2.1.