The biocatalytic transformations used by chemists are often restricted to simple functional-group interconversions. In contrast, nature has developed complexity-generating biocatalytic reactions within natural product pathways. These sophisticated catalysts are rarely employed by chemists, because the substrate scope, selectivity and robustness of these catalysts are unknown. Our strategy to bridge the gap between the biosynthesis and synthetic chemistry communities leverages the diversity of catalysts available within natural product pathways. Here we show that, starting from a suite of biosynthetic enzymes, catalysts with complementary substrate scope as well as selectivity can be identified. This strategy has been applied to the oxidative dearomatization of phenols, a chemical transformation that rapidly builds molecular complexity from simple starting materials and cannot be accomplished with high selectivity using existing catalytic methods. Using enzymes from biosynthetic pathways, we have successfully developed a method to produce ortho-quinol products with controlled site- and stereoselectivity. Furthermore, we have capitalized on the scalability and robustness of this method in gram-scale reactions as well as multi-enzyme and chemoenzymatic cascades.
The remarkable degree of synthetic selectivity found in Nature is exemplified by the biosynthesis of paralytic shellfish toxins such as saxitoxin. The polycyclic core shared by saxitoxin and its relatives is assembled and subsequently elaborated through the installation of hydroxyl groups with exquisite precision that is not possible to replicate with traditional synthetic methods. Here, we report the identification of the enzymes that carry out a subset of C-H functionalizations involved in paralytic shellfish toxin biosynthesis. We have shown that three Rieske oxygenases mediate hydroxylation reactions with perfect site- and stereoselectivity. Specifically, the Rieske oxygenase SxtT is responsible for selective hydroxylation of a tricyclic precursor to the famous natural product saxitoxin, and a second Rieske oxygenase, GxtA, selectively hydroxylates saxitoxin to access the oxidation pattern present in gonyautoxin natural products. Unexpectedly, a third Rieske oxygenase, SxtH, does not hydroxylate tricyclic intermediates, but rather a linear substrate prior to tricycle formation, rewriting the biosynthetic route to paralytic shellfish toxins. Characterization of SxtT, SxtH, and GxtA is the first demonstration of enzymes carrying out C-H hydroxylation reactions in paralytic shellfish toxin biosynthesis. Additionally, the reactions of these oxygenases with a suite of saxitoxin-related molecules are reported, highlighting the substrate promiscuity of these catalysts and the potential for their application in the synthesis of natural and unnatural saxitoxin congeners.
Biocatalysts that perform C-H hydroxylation exhibit exceptional substrate specificity and site-selectivity, often through the use of high valent oxidants to activate these inert bonds. Rieske oxygenases are examples of enzymes with the ability to perform precise mono-or dioxygenation reactions on a variety of substrates. Understanding the structural features of Rieske oxygenases responsible for control over selectivity is essential to enable the development of this class of enzymes for biocatalytic applications. Decades of research has illuminated the critical features common to Rieske oxygenases, however, structural information for enzymes that functionalize diverse scaffolds is limited. Here, we report the structures of two Rieske monooxygenases involved in the biosynthesis of paralytic shellfish toxins (PSTs), SxtT and GxtA, adding to the short list of structurally characterized Rieske oxygenases. Based on these structures, substrate-bound structures, and mutagenesis experiments, we implicate specific residues in substrate positioning and the divergent reaction selectivity observed in these two enzymes.
Rieske oxygenases exploit the reactivity of iron to perform chemically challenging C–H bond functionalization reactions. Thus far, only a handful of Rieske oxygenases have been structurally characterized and remarkably little information exists regarding how these enzymes use a common architecture and set of metallocenters to facilitate a diverse range of reactions. Herein, we detail how two Rieske oxygenases SxtT and GxtA use different protein regions to influence the site-selectivity of their catalyzed monohydroxylation reactions. We present high resolution crystal structures of SxtT and GxtA with the native β-saxitoxinol and saxitoxin substrates bound in addition to a Xenon-pressurized structure of GxtA that reveals the location of a substrate access tunnel to the active site. Ultimately, this structural information allowed for the identification of six residues distributed between three regions of SxtT that together control the selectivity of the C–H hydroxylation event. Substitution of these residues produces a SxtT variant that is fully adapted to exhibit the non-native site-selectivity and substrate scope of GxtA. Importantly, we also found that these selectivity regions are conserved in other structurally characterized Rieske oxygenases, providing a framework for predictively repurposing and manipulating Rieske oxygenases as biocatalysts.
Small molecules that bind to voltage-gated sodium channels (VGSCs) are promising leads in the treatment of numerous neurodegenerative diseases and pain. Nature is a highly skilled medicinal chemist in this regard, designing potent VGSC ligands capable of binding to and blocking the channel, thereby offering compounds of potential therapeutic interest. Paralytic shellfish toxins (PSTs), produced by cyanobacteria and marine dinoflagellates, are examples of these naturally occurring small molecule VGSC blockers that can potentially be leveraged to solve human health concerns. Unfortunately, the remarkable potency of these natural products results in equally exceptional toxicity, presenting a significant challenge for the therapeutic application of these compounds. Identifying less potent analogs and convenient methods for accessing them therefore provides an attractive approach to developing molecules with enhanced therapeutic potential. Fortunately, Nature has evolved tools to modulate the toxicity of PSTs through selective hydroxylation, sulfation, and desulfation of the core scaffold. Here, we demonstrate the function of enzymes encoded in cyanobacterial PST biosynthetic gene clusters that have evolved specifically for the sulfation of highly functionalized PSTs, the substrate scope of these enzymes, and elucidate the biosynthetic route from saxitoxin to monosulfated gonyautoxins and disulfated C-toxins. Finally, the binding affinities of the nonsulfated, monosulfated, and disulfated products of these enzymatic reactions have been evaluated for VGSC binding affinity using mouse whole brain membrane preparations to provide an assessment of relative toxicity. These data demonstrate the unique detoxification effect of sulfotransferases in PST biosynthesis, providing a potential mechanism for the development of more attractive PST-derived therapeutic analogs.
Aetokthonotoxin (AETX) is a cyanobacterial neurotoxin that causes vacuolar myelinopathy, a neurological disease that is particularly deadly to bald eagles in the United States. The recently characterized AETX is structurally unique among cyanotoxins and is composed of a pentabrominated biindole nitrile. Herein we report the discovery of an efficient, five-enzyme biosynthetic pathway that the freshwater cyanobacterium Aetokthonos hydrillicola uses to convert two molecules of tryptophan to AETX. We demonstrate that the biosynthetic pathway follows a convergent route in which two functionalized indole monomers are assembled and then reunited by biaryl coupling catalyzed by the cytochrome P450 AetB. Our results revealed enzymes with novel biochemical functions, including the single-component flavin-dependent tryptophan halogenase AetF and the iron-dependent nitrile synthase AetD.
Biocatalytic reactions embody many features of ideal chemical transformations, including the potential for impeccable selectivity, high catalytic efficiency, mild reaction conditions and the use of environmentally benign reagents. These advantages have created a demand for biocatalysts that expand the portfolio of complexity-generating reactions available to synthetic chemists. However, the tradeoff that often exists between the substrate scope of a biocatalyst and its selectivity limits the application of enzymes in synthesis. We recently demonstrated that a flavin-dependent monooxygenase, TropB, maintains high levels of site-and stereoselectivity across a range of structurally diverse substrates. Herein, we disclose the structural basis for substrate binding in TropB, which performs a synthetically challenging asymmetric oxidative dearomatization reaction with exquisite site-and stereoselectivity across a range of phenol substrates, providing a foundation for future protein engineering and reaction development efforts. Our hypothesis for substrate binding is informed by a crystal structure of TropB and molecular dynamics simulations with the corresponding computational TropB model and is supported by experimental data. In contrast to canonical class A FAD-dependent monooxygenases in which substrates bind in a protonated form, our data indicate that the phenolate form of the substrate binds in the active site. Furthermore, the substrate position is controlled through twopoint binding of the phenolate oxygen to Arg206 and Tyr239, which are shown to have distinct and essential roles in catalysis. Arg206 is involved in the reduction of the flavin cofactor, suggesting a role in flavin dynamics. Further, QM/MM simulations reveal the interactions that govern the facial selectivity that leads to a highly enantioselective transformation. Thus, the structural origins of the high levels of site-and stereoselectivity observed in reactions of TropB across a range of substrates are elucidated, providing a foundation for future protein engineering and reaction development efforts.
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