Louise K. Charkoudian

Expanding the Fluorine Chemistry of Living Systems Using Engineered Polyketide Synthase Pathways

Stitching in Fluoroacetate Polyketide synthase enzymes stitch together an impressively diverse series of organic compounds from simple acetate and propionate building blocks. Walker et al. (p. 1089) now show that these biochemical pathways can be engineered to incorporate fluoroacetate—a primary product of the only known native enzymatic fluorination route—into tri- and tetraketides. In Escherichia coli cells, this process shows potential as a versatile means of inserting fluorine substituents into a range of complex molecules for use in pharmaceutical and agrochemical research. Biochemical pathways can be engineered to incorporate fluoroactetate into tri- and tetraketides in place of acetate. Organofluorines represent a rapidly expanding proportion of molecules that are used in pharmaceuticals, diagnostics, agrochemicals, and materials. Despite the prevalence of fluorine in synthetic compounds, the known biological scope is limited to a single pathway that produces fluoroacetate. Here, we demonstrate that this pathway can be exploited as a source of fluorinated building blocks for introduction of fluorine into natural-product scaffolds. Specifically, we have constructed pathways involving two polyketide synthase systems, and we show that fluoroacetate can be used to incorporate fluorine into the polyketide backbone in vitro. We further show that fluorine can be inserted site-selectively and introduced into polyketide products in vivo. These results highlight the prospects for the production of complex fluorinated natural products using synthetic biology.

Probing the interactions of an acyl carrier protein domain from the 6-deoxyerythronolide B synthase

The assembly‐line architecture of polyketide synthases (PKSs) provides an opportunity to rationally reprogram polyketide biosynthetic pathways to produce novel antibiotics. A fundamental challenge toward this goal is to identify the factors that control the unidirectional channeling of reactive biosynthetic intermediates through these enzymatic assembly lines. Within the catalytic cycle of every PKS module, the acyl carrier protein (ACP) first collaborates with the ketosynthase (KS) domain of the paired subunit in its own homodimeric module so as to elongate the growing polyketide chain and then with the KS domain of the next module to translocate the newly elongated polyketide chain. Using NMR spectroscopy, we investigated the features of a structurally characterized ACP domain of the 6‐deoxyerythronolide B synthase that contribute to its association with its KS translocation partner. Not only were we able to visualize selective protein–protein interactions between the two partners, but also we detected a significant influence of the acyl chain substrate on this interaction. A novel reagent, CF3‐S‐ACP, was developed as a 19F NMR spectroscopic probe of protein–protein interactions. The implications of our findings for understanding intermodular chain translocation are discussed.

Iron prochelator BSIH protects retinal pigment epithelial cells against cell death induced by hydrogen peroxide

Dysregulation of localized iron homeostasis is implicated in several degenerative diseases, including Parkinsons, Alzheimers, and age-related macular degeneration, wherein iron-mediated oxidative stress is hypothesized to contribute to cell death. Inhibiting toxic iron without altering normal metal-dependent processes presents significant challenges for standard small molecule chelating agents. We previously introduced BSIH (isonicotinic acid [2-(4,4,5,5-tetramethyl-[1,3,2]dioxaborolan-2-yl)-benzylidene]-hydrazide) prochelators that are converted by hydrogen peroxide into SIH (salicylaldehyde isonicotinoyl hydrazone) chelating agents that inhibit iron-catalyzed hydroxyl radical generation. Here, we show that BSIH protects a cultured cell model for retinal pigment epithelium against cell death induced by hydrogen peroxide. BSIH is more stable than SIH in cell culture medium and is more protective during long-term experiments. Repetitive exposure of cells to BSIH is nontoxic, whereas SIH and desferrioxamine induce cell death after repeated exposure. Combined, our results indicate that cell protection by BSIH involves iron sequestration that occurs only when the cells are stressed by hydrogen peroxide. These findings suggest that prochelators discriminate toxic iron from healthy iron and are promising candidates for neuro- and retinal protection.

New Structural Data Reveal the Motion of Carrier Proteins in Nonribosomal Peptide Synthesis.

Abstract The nonribosomal peptide synthetases (NRPSs) are one of the most promising resources for the production of new bioactive molecules. The mechanism of NRPS catalysis is based around sequential catalytic domains: these are organized into modules, where each module selects, modifies, and incorporates an amino acid into the growing peptide. The intermediates formed during NRPS catalysis are delivered between enzyme centers by peptidyl carrier protein (PCP) domains, which makes PCP interactions and movements crucial to NRPS mechanism. PCP movement has been linked to the domain alternation cycle of adenylation (A) domains, and recent complete NRPS module structures provide support for this hypothesis. However, it appears as though the A domain alternation alone is insufficient to account for the complete NRPS catalytic cycle and that the loaded state of the PCP must also play a role in choreographing catalysis in these complex and fascinating molecular machines.

Modifications of boronic ester pro-chelators triggered by hydrogen peroxide tune reactivity to inhibit metal-promoted oxidative stress

Several new analogs of salicylaldehyde isonicotinoyl hydrazone (SIH) and salicylaldehyde benzoyl hydrazone (SBH) that contain an aryl boronic ester (BSIH, BSBH) or acid (BASIH) in place of an aryl hydroxide have been synthesized and characterized as masked metal ion chelators. These pro-chelators show negligible interaction with iron(III), although the boronic acid versions exhibit some interaction with copper(II), zinc(II) and nickel(II). Hydrogen peroxide oxidizes the aryl boronate to phenol, thus converting the pro-chelators to tridentate ligands with high affinity metal binding properties. An X-ray crystal structure of a bis-ligated iron(III) complex, [Fe(SBH(m-OMe)(3))(2)]NO(3), confirms the meridonal binding mode of these ligands. Modifications of the aroyl ring of the chelators tune their iron affinity, whereas modifications on the boron-containing ring of the pro-chelators attenuate their reaction rates with hydrogen peroxide. Thus, the methoxy derivative pro-chelator (p-OMe)BASIH reacts with hydrogen peroxide nearly 5 times faster than the chloro derivative (m-Cl)BASIH. Both the rate of pro-chelator to chelator conversion as well as the metal binding affinity of the chelator influence the overall ability of these molecules to inhibit hydroxyl radical formation catalyzed by iron or copper in the presence of hydrogen peroxide and ascorbic acid. This pro-chelator strategy has the potential to improve the efficacy of medicinal chelators for inhibiting metal-promoted oxidative stress.

Probing the phosphopantetheine arm conformations of acyl carrier proteins using vibrational spectroscopy.

Acyl carrier proteins (ACPs) are universal and highly conserved domains central to both fatty acid and polyketide biosynthesis. These proteins tether reactive acyl intermediates with a swinging 4′-phosphopantetheine (Ppant) arm and interact with a suite of catalytic partners during chain transport and elongation while stabilizing the growing chain throughout the biosynthetic pathway. The flexible nature of the Ppant arm and the transient nature of ACP–enzyme interactions impose a major obstacle to obtaining structural information relevant to understanding polyketide and fatty acid biosynthesis. To overcome this challenge, we installed a thiocyanate vibrational spectroscopic probe on the terminal thiol of the ACP Ppant arm. This site-specific probe successfully reported on the local environment of the Ppant arm of two ACPs previously characterized by solution NMR, and was used to determine the solution exposure of the Ppant arm of an ACP from 6-deoxyerythronolide B synthase (DEBS). Given the sensitivity of the probe’s CN stretching band to conformational distributions resolved on the picosecond time scale, this work lays a foundation for observing the dynamic action-related structural changes of ACPs using vibrational spectroscopy.

Evolution of chemical diversity by coordinated gene swaps in type II polyketide gene clusters

Significance Type II polyketide natural products are powerful antimicrobial agents that are biosynthesized within bacteria by enzyme-encoding clusters of genes. We present a method to elucidate the evolution of these gene clusters as a whole, illuminating how natural selection has led to the chemical diversity of type II polyketides. Our approach can be applied to understand how other natural product gene clusters evolve. This understanding may aid efforts to access novel natural products and to design rational enzyme assemblies that produce chemicals of desired structures and activities. Natural product biosynthetic pathways generate molecules of enormous structural complexity and exquisitely tuned biological activities. Studies of natural products have led to the discovery of many pharmaceutical agents, particularly antibiotics. Attempts to harness the catalytic prowess of biosynthetic enzyme systems, for both compound discovery and engineering, have been limited by a poor understanding of the evolution of the underlying gene clusters. We developed an approach to study the evolution of biosynthetic genes on a cluster-wide scale, integrating pairwise gene coevolution information with large-scale phylogenetic analysis. We used this method to infer the evolution of type II polyketide gene clusters, tracing the path of evolution from the single ancestor to those gene clusters surviving today. We identified 10 key gene types in these clusters, most of which were swapped in from existing cellular processes and subsequently specialized. The ancestral type II polyketide gene cluster likely comprised a core set of five genes, a roster that expanded and contracted throughout evolution. A key C24 ancestor diversified into major classes of longer and shorter chain length systems, from which a C20 ancestor gave rise to the majority of characterized type II polyketide antibiotics. Our findings reveal that (i) type II polyketide structure is predictable from its gene roster, (ii) only certain gene combinations are compatible, and (iii) gene swaps were likely a key to evolution of chemical diversity. The lessons learned about how natural selection drives polyketide chemical innovation can be applied to the rational design and guided discovery of chemicals with desired structures and properties.

In Living Color: Bacterial Pigments as an Untapped Resource in the Classroom and Beyond

Recent advances in the study of natural products made by bacteria have laid the foundation for engineering these molecules and for developing cost-effective ways to manufacture them. In our lab, we study a number of natural products that are synthesized by harmless soil bacteria of the Streptomyces genus. Whereas our primary interest in these molecules is due to their antibiotic properties, many of these natural products have distinct colors [1]. (The reasons for why Streptomyces make antibiotics or pigments remain mysterious.) This article is intended to make the case to the scientific and educational communities that Streptomyces-derived natural products are an untapped source of useful biopigments. By sharing some of our own experiences in harnessing these pigments to create paint and paintings, we also hope to inspire others to explore the potential of Streptomyces-derived pigments in art, industry, and perhaps most importantly, the classroom. The pedagogical value of bacterial pigments is highlighted by the wide range of concepts and methods in chemistry, biology, and art that can be introduced to students in this context (see Box 1). Teachers can incorporate bacterial pigments into their lessons while introducing fundamental scientific principles ranging from the physics of color to the chemistry behind paints that fade in sunlight. Painting with living bacteria (Box 2) or extracting pigments from bacterial cultures (Box 3) provides a visual and kinesthetic activity to support key aspects of scientific investigations and methods learned in the classroom. Because the methods to do so are safe, inexpensive, and easily implementable in the everyday world, it is possible to use biopigments as a vehicle to introduce school children to science via art and vice versa. While many of these concepts and techniques are appropriate for the advanced high school or undergraduate classroom, even elementary school children can use bacterial paints prepared by their teacher to create art, an activity that may teach children at a young age that bacteria are a source of valuable materials rather than merely agents of disease. Box 1: Concepts at a Glance Leads into chemistry, microbiology, and biotechnology Chemical composition of paint (solubility and states of matter)¥, ‡ Structures of pigment molecules (electromagnetic radiation, electron configuration, valence bonds, molecular orbital theory)‡ Culturing Streptomyces and extracting their pigments (sterile culture techniques, natural product extraction techniques, solubility)‡ Painting Streptomyces on agar plates (bacterial growth control)¥,‡ Engineering bacteria to make new pigments (metabolic engineering of microbial systems)‡ Scaling up the production of bacterial pigments (large scale bioprocessing techniques, recombinant DNA technology)‡ UV absorber and radical scavengers as additives to paints (chemical structure and reactivity, radical reactions)‡ Leads into fine arts The perception of color (electromagnetic radiation, the eye as a spectrometer)¥, ‡ Paint constituents (pigments, binders, solvents, surfactants, additives)‡ Sources of pigments‡ Making paints from pigments (grinding pigments, suspending in binder)¥, ‡ History of pigments (art history)*, ¥, ‡ Fun stuff Drawing on paper with bacteria-derived paint*, ¥, ‡ Creating living art by painting with bacteria on agar medium*, ¥, ‡ ‡ = for undergraduate or advanced placement high school courses; ¥ = for high school courses; * = for elementary school courses

Probing the selectivity of β-hydroxylation reactions in non-ribosomal peptide synthesis using analytical ultracentrifugation

Bacteria and fungi use non-ribosomal peptide synthetases (NRPSs) to produce peptides of broad structural diversity and biological activity, many of which have proven to be of great importance for human health. The impressive diversity of non-ribosomal peptides originates in part from the action of tailoring enzymes that modify the structures of single amino acids and/or the mature peptide. Studying the interplay between tailoring enzymes and the peptidyl carrier proteins (PCPs) that anchor the substrates is challenging owing to the transient and complex nature of the protein-protein interactions. Using sedimentation velocity (SV) methods, we studied the collaboration between the PCPs and cytochrome P450 enzyme that results in the installation of β-hydroxylated amino acid precursors in the biosynthesis of the depsipeptide skyllamycin. We show that SV methods developed for the analytical ultracentrifuge are ideally suited for a quantitative exploration of PCP-enzyme equilibrium interactions. Our results suggest that the PCP itself and the presence of substrate covalently tethered to the PCP together facilitate productive PCP-P450 interactions, thereby revealing one of natures intricate strategies for installing interesting functionalities using natural product synthetases.

Natural product inhibitors of glucose-6-phosphate translocase

Inhibitors of glucose-6-phosphate translocase (G6P T1) control aberrant glucose levels and show promise as anticancer and anti-diabetic agents. This mini-review provides a concise overview of natural product and natural product analogs that inhibit G6P T1. The discovery and development of these inhibitors, as well as their efficacy in cell-based assays and in vivo, are discussed. Finally, we acknowledge the need for improved G6P T1 inhibitors and the potential to harness the programmable chemistry of microorganisms to meet this need.