Polyketide chain length control by chain length factor

Bacterial aromatic polyketides are pharmacologically important natural products. A critical parameter that dictates product structure is the carbon chain length of the polyketide backbone. Systematic manipulation of polyketide chain length represents a major unmet challenge in natural product biosynthesis. Polyketide chain elongation is catalyzed by a heterodimeric ketosynthase. In contrast to homodimeric ketosynthases found in fatty acid synthases, the active site cysteine is absent from the one subunit of this heterodimer. The precise role of this catalytically silent subunit has been debated over the past decade. We demonstrate here that this subunit is the primary determinant of polyketide chain length, thereby validating its designation as chain length factor. Using structure-based mutagenesis, we identified key residues in the chain length factor that could be manipulated to convert an octaketide synthase into a decaketide synthase and vice versa. These results should lead to novel strategies for the engineered biosynthesis of hitherto unidentified polyketide scaffolds.     

Successive complexometric determination of thorium and uranium in sulphuric acid media

A selective analytical extraction method for rapid successive complexometric determination of thorium(IV) and uranium(VI) in sulphuric acid media is described. The method is based on the extraction of thorium and uranium from sulphuric acid media with N-butylaniline or N-benzylaniline in chloroform. Both thorium and uranium are selectively and quantitatively extracted in the presence of ascorbic acid and EDTA. Most cations and anions do not interfere. The reduction of uranium(VI) with sodium dithionite at room temperature is rapid and quantitative and superior to that with ascorbic acid, which reduces uranium(VI) in boiling solution. The method is simple, rapid and accurate, and the experimental conditions are not highly critical.     

Assessing the balance between protein-protein interactions and enzyme-substrate interactions in the channeling of intermediates between polyketide synthase modules.

6-Deoxyerythronolide B synthase (DEBS) is the modular polyketide synthase (PKS) that catalyzes the biosynthesis of 6-deoxyerythronolide B (6-dEB), the aglycon precursor of the antibiotic erythromycin. The biosynthesis of 6-dEB exemplifies the extraordinary substrate- and stereo-selectivity of this family of multifunctional enzymes. Paradoxically, DEBS has been shown to be an attractive scaffold for combinatorial biosynthesis, indicating that its constituent modules are also very tolerant of unnatural substrates. By interrogating individual modules of DEBS with a panel of diketides activated as N-acetylcysteamine (NAC) thioesters, it was recently shown that individual modules have a marked ability to discriminate among certain diastereomeric diketides. However, since free NAC thioesters were used as substrates in these studies, the modules were primed by a diffusive process, which precluded involvement of the covalent, substrate-channeling mechanism by which enzyme-bound intermediates are directly transferred from one module to the next in a multimodular PKS. Recent evidence pointing to a pivotal role for protein-protein interactions in the substrate-channeling mechanism has prompted us to develop novel assays to reassess the steady-state kinetic parameters of individual DEBS modules when primed in a more "natural" channeling mode by the same panel of diketide substrates used earlier. Here we describe these assays and use them to quantify the kinetic benefit of linker-mediated substrate channeling in a modular PKS. This benefit can be substantial, especially for intrinsically poor substrates. Examples are presented where the k(cat) of a module for a given diketide substrate increases >100-fold when the substrate is presented to the module in a channeling mode as opposed to a diffusive mode. However, the substrate specificity profiles for individual modules are conserved regardless of the mode of presentation. By highlighting how substrate channeling can allow PKS modules to effectively accept and process intrinsically poor substrates, these studies provide a rational basis for examining the enormous untapped potential for combinatorial biosynthesis via module rearrangement.     

Polyketide double bond biosynthesis. Mechanistic analysis of the dehydratase-containing module 2 of the picromycin/methymycin polyketide synthase

Picromycin/methymycin synthase (PICS) is a modular polyketide synthase (PKS) that is responsible for the biosynthesis of both 10-deoxymethynolide (1) and narbonolide (2), the parent 12- and 14-membered aglycone precursors of the macrolide antibiotics methymycin and picromycin, respectively. PICS module 2 is a dehydratase (DH)-containing module that catalyzes the formation of the unsaturated triketide intermediate using malonyl-CoA as the chain extension substrate. Recombinant PICS module 2+TE, with the PICS thioesterase domain appended to the C-terminus to allow release of polyketide products, was expressed in Escherichia coli. Purified PICS module 2+TE converted malonyl-CoA and 4, the N-acetylcysteamine thioester of (2S,3R)-2-methyl-3-hydroxypentanoic acid, to a 1:2 mixture of the triketide acid (4S,5R)-4-methyl-5-hydroxy-2-heptenoic acid (5) and (3S,4S,5R)-3,5-dihydroxy-4-methyl-n-heptanoic acid-delta-lactone (10) with a combined kcat of 0.6 min(-1). The triketide lactone 10 is formed by thioesterase-catalyzed cyclization of the corresponding d-3-hydroxyacyl-SACP intermediate, a reaction which competes with dehydration catalyzed by the dehydratase domain. PICS module 2+TE showed a strong preference for the syn-diketide-SNAC 4, with a 20-fold greater kcat/K(m) than the anti-(2S,3S)-diketide-SNAC 14, and a 40-fold advantage over the syn-(2R,3S)-diketide-SNAC 13. PICS module 2(DH(0))+TE, with an inactivated DH domain, produced exclusively 10, while three PICS module 2(KR(0))+TE mutants, with inactivated KR domains, produced exclusively or predominantly the unreduced triketide ketolactone, (4S,5R)-3-oxo-4-methyl-5-hydroxy-n-heptanoic acid-delta-lactone (7). These studies establish for the first time the structure and stereochemistry of the intermediates of a polyketide chain elongation cycle catalyzed by a DH-containing module, while confirming the importance of key active site residues in both KR and DH domains.     

Combined experimental and computational study of W(II), Ru(II), Pt(IV) and Cu(I) amine and amido complexes using 15N NMR spectroscopy

Using 2D proton-coupled gHSQC pulse sequences in addition to 1D ¹⁵N NMR experiments of ¹⁵N labeled systems, ¹⁵N NMR chemical shifts of a range of transition metal amido and amine complexes were determined. Tungsten(II), ruthenium(II), platinum(IV) and copper(I) complexes with aniline and their anilido variants were studied and compared to free aniline, lithium anilido and anilinium tetrafluoroborate. Upon coordination of aniline to transition metals, upfield chemical shifts of 20–60 ppm were observed. Deprotonation of the amine complexes to form amido complexes resulted in downfield chemical shifts of 40–60 ppm for all of the complexes except for the tungsten d⁴ system. For the tungsten(II) complexes, the cationic aniline complex displayed a downfield shift of approximately 56 ppm relative to the neutral anilido complex. The change in chemical shift for amine to amido conversion is proposed to depend on the ability of the amido ligand to π-bond with the metal center, which influences the magnitude of the paramagnetic screening term.     

Heterologous expression, purification, refolding, and structural-functional characterization of EP-B2, a self-activating barley cysteine endoprotease

We describe the heterologous expression in Escherichia coli of the proenzyme precursor to EP-B2, a cysteine endoprotease from germinating barley seeds. High yields (50 mg/l) of recombinant proEP-B2 were obtained from E. coli inclusion bodies in shake flask cultures following purification and refolding. The zymogen was rapidly autoactivated to its mature form under acidic conditions at a rate independent of proEP-B2 concentration, suggesting a cis mechanism of autoactivation. Mature EP-B2 was stable and active over a wide pH range and efficiently hydrolyzed a recombinant wheat gluten protein, alpha2-gliadin, at sequences with known immunotoxicity in celiac sprue patients. The X-ray crystal structure of mature EP-B2 bound to leupeptin was solved to 2.2 A resolution and provided atomic insights into the observed subsite specificity of the endoprotease. Our findings suggest that orally administered proEP-B2 may be especially well suited for treatment of celiac sprue.     

Precursor-directed biosynthesis of epothilone in Escherichia coli

Engineered biosynthetic pathways provide a powerful method for generating complex molecules. Precursor-directed biosynthesis, which combines chemical synthesis and enzymatic transformations, allows non-native starting materials to be incorporated into biosynthetic pathways. Using this approach, we achieved the production of the anticancer agent epothilone C in Escherichia coli. An E. coli strain was engineered to express the last three modules of the epothilone biosynthetic pathway (epoD-M6, epoE, and epoF) and the substrate required to complement the biosynthetic enzymes was obtained by chemical synthesis. Under high-density cell culture conditions, the E. coli strain processed exogenously fed synthetic substrate into epothilone C at levels comparable to the native host (1 mg/L) and at higher levels than other heterologous hosts. Importantly, this precursor-directed approach will allow chemical modifications to be introduced into the polyketide backbone and may ultimately provide access to epothilone analogues with improved pharmacological properties in quantities sufficient for clinical development.     

Solvent extraction and separation of gallium, indium and thallium with N-benzylaniline

A simple and rapid method is proposed for the separation of tervalent gallium, indium and thallium by solvent extraction with N-benzylaniline in chloroform from different concentrations of hydrochloric acid. Thallium and gallium are extracted from 1M and 7.0-7.5M hydrochloric acid respectively. Indium is finally extracted from hydriodic acid. These metals in the final extracts are determined complexometrically. Interference from some cations can easily be eliminated by reduction with sulphite, followed by selective oxidation of thallium(I) to thallium(III) with saturated bromine water, and from others by the use of thioglycollic acid as a masking agent in the extraction of gallium and indium. Most common anions cause no interference. Log-log plots of distribution coefficients vs. concentration of amine for gallium, indium and thallium indicate a 2:1 limiting mole ratio of amine to these metals.     

Time-reversing array retrofocusing in noisy environments

Acoustic time reversal is a robust means of retrofocusing acoustic energy, in both time and space, to the original sound-source location. However, noise may limit the performance of a time-reversing array (TRA) at long source-array ranges, or when the original-source or TRA-element power levels are low. The operation of a TRA requires two steps (reception and transmission) so both TRA-broadcast noise and ambient noise must be taken into account. In this paper, predictions are made for how a simple omnidirectional noise field influences the probability that the signal amplitude from a narrow-band TRA will exceed the noise at the TRA's retrofocus. A general formulation for the probability of TRA retrofocusing, which can be used for TRA design, is developed that includes: the variance of the noise field, the original source strength, the TRA's element output power, the number of TRA elements (N), and the propagation characteristics of the environment. This formulation predicts that a TRA's array gain (in dB) at the retrofocus may be as high as + 10log10(N) to + 20 log10(N) depending on the relative strengths of the original source and the TRA's elements. Monte Carlo simulations in both a free-space environment and a shallow-ocean sound-channel environment compare well to this probability formulation even when simple approximate parametric relationships for the appropriate Green's functions are used. The dominant deviation between theory and simulation in the sound channel is caused by acoustic absorption.     

Resolving Multiple Protein–Peptide Binding Events: Implication for HLA-DQ2 Mediated Antigen Presentation in Celiac Disease

Techniques that can effectively separate protein–peptide complexes from free peptides have shown great value in major histocompatibility complex (MHC)–peptide binding studies. However, most of the available techniques are limited to measuring the binding of a single peptide to an MHC molecule. As antigen presentation in vivo involves both endogenous ligands and exogenous antigens, the deconvolution of multiple binding events necessitates the implementation of a more powerful technique. Here we show that capillary electrophoresis coupled to fluorescence detection (CE–FL) can resolve multiple MHC–peptide binding events owing to its superior resolution and the ability to simultaneously monitor multiple emission channels. We utilized CE–FL to investigate competition and displacement of endogenous peptides by an immunogenic gluten peptide for binding to HLA-DQ2. Remarkably, this immunogenic peptide could displace CLIP peptides from the DQ2 binding site at neutral but not acidic pH. This unusual ability of the gluten peptide supports a direct loading mechanism of antigen presentation in extracellular environment, a property that could explain the antigenicity of dietary gluten in celiac disease.