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Prenyltransfer is an early-stage carbon-hydrogen bond (C-H) functionalization prevalent in the biosynthesis of a diverse array of biologically active bacterial, fungal, plant, and metazoan diketopiperazine (DKP) alkaloids. Toward the development of a unified strategy for biocatalytic construction of prenylated DKP indole alkaloids, we sought to identify and characterize a substrate-permissive C2 reverse prenyltransferase (PT). As the first tailoring event within the biosynthesis of cytotoxic notoamide metabolites, PT NotF catalyzes C2 reverse prenyltransfer of brevianamide F. Solving a crystal structure of NotF (in complex with native substrate and prenyl donor mimic dimethylallyl S-thiolodiphosphate (DMSPP)) revealed a large, solvent-exposed active site, intimating NotF may possess a significantly broad substrate scope. To assess the substrate selectivity of NotF, we synthesized a panel of 30 sterically and electronically differentiated tryptophanyl DKPs, the majority of which were selectively prenylated by NotF in synthetically useful conversions (2 to >99%). Quantitative representation of this substrate library and development of a descriptive statistical model provided insight into the molecular origins of NotF's substrate promiscuity. This approach enabled the identification of key substrate descriptors (electrophilicity, size, and flexibility) that govern the rate of NotF-catalyzed prenyltransfer, and the development of an "induced fit docking (IFD)-guided" engineering strategy for improved turnover of our largest substrates. We further demonstrated the utility of NotF in tandem with oxidative cyclization using flavin monooxygenase, BvnB. This one-pot, in vitro biocatalytic cascade enabled the first chemoenzymatic synthesis of the marine fungal natural product, (-)-eurotiumin A, in three steps and 60% overall yield.

Medically important flaviviruses cause diverse disease pathologies and collectively are responsible for a major global disease burden. A contributing factor to pathogenesis is secreted flavivirus nonstructural protein 1 (NS1). Despite demonstrated protection by NS1-specific antibodies against lethal flavivirus challenge, the structural and mechanistic basis remains unknown. Here, we present three crystal structures of full-length dengue virus NS1 complexed with a flavivirus-cross-reactive, NS1-specific monoclonal antibody, 2B7, at resolutions between 2.89 and 3.96 angstroms. These structures reveal a protective mechanism by which two domains of NS1 are antagonized simultaneously. The NS1 wing domain mediates cell binding, whereas the β-ladder triggers downstream events, both of which are required for dengue, Zika, and West Nile virus NS1-mediated endothelial dysfunction. These observations provide a mechanistic explanation for 2B7 protection against NS1-induced pathology and demonstrate the potential of one antibody to treat infections by multiple flaviviruses.

The paraherquamides are potent anthelmintic natural products with complex heptacyclic scaffolds. One key feature of these molecules is the spiro-oxindole moiety that lends a strained three-dimensional architecture to these structures. The flavin monooxygenase PhqK was found to catalyze spirocycle formation through two parallel pathways in the biosynthesis of paraherquamides A and G. Two new paraherquamides (K and L) were isolated from a ΔphqK strain of Penicillium simplicissimum, and subsequent enzymatic reactions with these compounds generated two additional metabolites, paraherquamides M and N. Crystal structures of PhqK in complex with various substrates provided a foundation for mechanistic analyses and computational studies. While it is evident that PhqK can react with various substrates, reaction kinetics and molecular dynamics simulations indicated that the dioxepin-containing paraherquamide L is the favored substrate. Through this effort, we have elucidated a key step in the biosynthesis of the paraherquamides and provided a rationale for the selective spirocyclization of these powerful anthelmintic agents.

Natural product biosynthetic pathways are replete with enzymes repurposed for new catalytic functions. In some modular polyketide synthase (PKS) pathways, a GCN5-related N-acetyltransferase (GNAT)-like enzyme with an additional decarboxylation function initiates biosynthesis. Here, we probe two PKS GNAT-like domains for the dual activities of S-acyl transfer from coenzyme A (CoA) to an acyl carrier protein (ACP) and decarboxylation. The GphF and CurA GNAT-like domains selectively decarboxylate substrates that yield the anticipated pathway starter units. The GphF enzyme lacks detectable acyl transfer activity, and a crystal structure with an isobutyryl-CoA product analog reveals a partially occluded acyltransfer acceptor site. Further analysis indicates that the CurA GNAT-like domain also catalyzes only decarboxylation, and the initial acyl transfer is catalyzed by an unidentified enzyme. Thus, PKS GNAT-like domains are re-classified as GNAT-like decarboxylases. Two other decarboxylases, malonyl-CoA decarboxylase and EryM, reside on distant nodes of the superfamily, illustrating the adaptability of the GNAT fold.

Prenylated indole alkaloids such as the calmodulin-inhibitory malbrancheamides and anthelmintic paraherquamides possess great structural diversity and pharmaceutical utility. Here, we report complete elucidation of the malbrancheamide biosynthetic pathway accomplished through complementary approaches. These include a biomimetic total synthesis to access the natural alkaloid and biosynthetic intermediates in racemic form and in vitro enzymatic reconstitution to provide access to the natural antipode (+)-malbrancheamide. Reductive cleavage of an L-Pro-L-Trp dipeptide from the MalG non-ribosomal peptide synthetase (NRPS) followed by reverse prenylation and a cascade of post-NRPS reactions culminates in an intramolecular [4+2] hetero-Diels-Alder (IMDA) cyclization to furnish the bicyclo[2.2.2]diazaoctane scaffold. Enzymatic assembly of optically pure (+)-premalbrancheamide involves an unexpected zwitterionic intermediate where MalC catalyses enantioselective cycloaddition as a bifunctional NADPH-dependent reductase/Diels-Alderase. The crystal structures of substrate and product complexes together with site-directed mutagenesis and molecular dynamics simulations demonstrate how MalC and PhqE (its homologue from the paraherquamide pathway) catalyse diastereo- and enantioselective cyclization in the construction of this important class of secondary metabolites.

Fatty acid biosynthesis in α- and γ-proteobacteria requires two functionally distinct dehydratases, FabA and FabZ. Here, mechanistic cross-linking facilitates the structural characterization of a stable hexameric complex of six Escherichia coli FabZ dehydratase subunits with six AcpP acyl carrier proteins. The crystal structure sheds light on the divergent substrate selectivity of FabA and FabZ by revealing distinct architectures of the binding pocket. Molecular dynamics simulations demonstrate differential biasing of substrate orientations and conformations within the active sites of FabA and FabZ such that FabZ is preorganized to catalyze only dehydration, while FabA is primed for both dehydration and isomerization.

Detecting viral infection is a key role of the innate immune system. The genomes of some RNA viruses have a high CpG dinucleotide content relative to most vertebrate cell RNAs, making CpGs a molecular marker of infection. The human zinc-finger antiviral protein (ZAP) recognizes CpG, mediates clearance of the foreign CpG-rich RNA, and causes attenuation of CpG-rich RNA viruses. While ZAP binds RNA, it lacks enzymatic activity that might be responsible for RNA degradation and thus requires interacting cofactors for its function. One of these cofactors, KHNYN, has a predicted nuclease domain. Using biochemical approaches, we found that the KHNYN NYN domain is a single-stranded RNA ribonuclease that does not have sequence specificity and digests RNA with or without CpG dinucleotides equivalently in vitro. We show that unlike most KH domains, the KHNYN KH domain does not bind RNA. Indeed, a crystal structure of the KH region revealed a double-KH domain with a negatively charged surface that accounts for the lack of RNA binding. Rather, the KHNYN C-terminal domain (CTD) interacts with the ZAP RNA-binding domain (RBD) to provide target RNA specificity. We define a minimal complex composed of the ZAP RBD and the KHNYN NYN-CTD and use a fluorescence polarization assay to propose a model for how this complex interacts with a CpG dinucleotide-containing RNA. In the context of the cell, this module would represent the minimum ZAP and KHNYN domains required for CpG-recognition and ribonuclease activity essential for attenuation of viruses with clusters of CpG dinucleotides. [ABSTRACT FROM AUTHOR]

Dozens of type A malyngamides, principally identified by a decorated six-membered cyclohexanone headgroup and methoxylated lyngbic acid tail, have been isolated over several decades. Their environmental sources include macro- and microbiotic organisms, including sea hares, red alga, and cyanobacterial assemblages, but the true producing organism has remained enigmatic. Many type A analogues display potent bioactivity in human-health related assays, spurring an interest in this molecular class and its biosynthetic pathway. Here, we present the discovery of the type A malyngamide biosynthetic pathway in the first sequenced genome of the cyanobacterial genus Okeania. Bioinformatic analysis of two cultured Okeania genome assemblies identified 62 and 68 kb polyketide synthase/nonribosomal peptide synthetase (PKS/NRPS) pathways with unusual loading and termination genes. NMR data of malyngamide C acetate derived from 13C-substrate-fed cultures provided evidence that an intact octanoate moiety is transferred to the first KS module via a LipM homologue originally associated with lipoic acid metabolism and implicated an inactive ketoreductase (KR0) as critical for six-membered ring formation, a hallmark of the malyngamide family. Phylogenetic analysis and homology modeling of the penultimate KR0 domain inferred structural cofactor binding and active site alterations as contributory to domain dysfunction, which was confirmed by recombinant protein expression and NADPH binding assay. The carbonyl retained from this KR0 ultimately enables an intramolecular Knoevenagel condensation to form the characteristic cyclohexanone ring. Understanding this critical step allows assignment of a biosynthetic model for all type A malyngamides, whereby well-characterized tailoring modifications explain the surprising proliferation and diversity of analogues.

Modular type I polyketide synthases (PKSs) produce some of the most chemically complex metabolites in nature through a series of multienzyme modules. Each module contains a variety of catalytic domains to selectively tailor the growing molecule. PKS O-methyltransferases ( O-MTs) are predicted to methylate β-hydroxyl or β-keto groups, but their activity and structure have not been reported. We determined the domain boundaries and characterized the catalytic activity and structure of the StiD and StiE O-MTs, which methylate opposite β-hydroxyl stereocenters in the myxobacterial stigmatellin biosynthetic pathway. Substrate stereospecificity was demonstrated for the StiD O-MT. Key catalytic residues were identified in the crystal structures and investigated in StiE O-MT via site-directed mutagenesis and further validated with the cyanobacterial CurL O-MT from the curacin biosynthetic pathway. Initial structural and biochemical analysis of PKS O-MTs supplies a new chemoenzymatic tool, with the unique ability to selectively modify hydroxyl groups during polyketide biosynthesis.

The unusual feature of a t-butyl group is found in several marine-derived natural products including apratoxin A, a Sec61 inhibitor produced by the cyanobacterium Moorea bouillonii PNG 5-198. Here, we determine that the apratoxin A t-butyl group is formed as a pivaloyl acyl carrier protein (ACP) by AprA, the polyketide synthase (PKS) loading module of the apratoxin A biosynthetic pathway. AprA contains an inactive "pseudo" GCN5-related N-acetyltransferase domain (ΨGNAT) flanked by two methyltransferase domains (MT1 and MT2) that differ distinctly in sequence. Structural, biochemical, and precursor incorporation studies reveal that MT2 catalyzes unusually coupled decarboxylation and methylation reactions to transform dimethylmalonyl-ACP, the product of MT1, to pivaloyl-ACP. Further, pivaloyl-ACP synthesis is primed by the fatty acid synthase malonyl acyltransferase (FabD), which compensates for the ΨGNAT and provides the initial acyl-transfer step to form AprA malonyl-ACP. Additionally, images of AprA from negative stain electron microscopy reveal multiple conformations that may facilitate the individual catalytic steps of the multienzyme module.

Hapalindole alkaloids are a structurally diverse class of cyanobacterial natural products defined by their varied polycyclic ring systems and diverse biological activities. These complex metabolites are generated from a common biosynthetic intermediate by the Stig cyclases in three mechanistic steps: a rare Cope rearrangement, 6-exo-trig cyclization, and electrophilic aromatic substitution. Here we report the structure of HpiC1, a Stig cyclase that catalyzes the formation of 12-epi-hapalindole U in vitro. The 1.5-Å structure revealed a dimeric assembly with two calcium ions per monomer and with the active sites located at the distal ends of the protein dimer. Mutational analysis and computational methods uncovered key residues for an acid-catalyzed [3,3]-sigmatropic rearrangement, as well as specific determinants that control the position of terminal electrophilic aromatic substitution, leading to a switch from hapalindole to fischerindole alkaloids.

Natural product biosynthetic pathways contain a plethora of enzymatic tools to carry out difficult biosynthetic transformations. Here, we discover an unusual mononuclear iron-dependent methyltransferase that acts in the initiation steps of apratoxin A biosynthesis (AprA MT1). Fe3+-replete AprA MT1 catalyzes one or two methyl transfer reactions on the substrate malonyl-ACP (acyl carrier protein), whereas Co2+, Fe2+, Mn2+, and Ni2+ support only a single methyl transfer. MT1 homologues exist within the "GNAT" (GCN5-related N-acetyltransferase) loading modules of several modular biosynthetic pathways with propionyl, isobutyryl, or pivaloyl starter units. GNAT domains are thought to catalyze decarboxylation of malonyl-CoA and acetyl transfer to a carrier protein. In AprA, the GNAT domain lacks both decarboxylation and acyl transfer activity. A crystal structure of the AprA MT1-GNAT di-domain with bound Mn2+, malonate, and the methyl donor S-adenosylmethionine (SAM) reveals that the malonyl substrate is a bidentate metal ligand, indicating that the metal acts as a Lewis acid to promote methylation of the malonyl α-carbon. The GNAT domain is truncated relative to functional homologues. These results afford an expanded understanding of MT1-GNAT structure and activity and permit the functional annotation of homologous GNAT loading modules both with and without methyltransferases, additionally revealing their rapid evolutionary adaptation in different biosynthetic contexts.

Malbrancheamide is a dichlorinated fungal indole alkaloid isolated from both Malbranchea aurantiaca and Malbranchea graminicola that belongs to a family of natural products containing a characteristic bicyclo[2.2.2]diazaoctane core. The introduction of chlorine atoms on the indole ring of malbrancheamide differentiates it from other members of this family and contributes significantly to its biological activity. In this study, we characterized the two flavin-dependent halogenases involved in the late-stage halogenation of malbrancheamide in two different fungal strains. MalA and MalA' catalyze the iterative dichlorination and monobromination of the free substrate premalbrancheamide as the final steps in the malbrancheamide biosynthetic pathway. Two unnatural bromo-chloro-malbrancheamide analogues were generated through MalA-mediated chemoenzymatic synthesis. Structural analysis and computational studies of MalA' in complex with three substrates revealed that the enzyme represents a new class of zinc-binding flavin-dependent halogenases and provides new insights into a potentially unique reaction mechanism.

A key challenge in the SAD phasing method is solving a structure when the anomalous signal-to-noise ratio is low. A simple theoretical framework for describing measurements of anomalous differences and the resulting useful anomalous correlation and anomalous signal in a SAD experiment is presented. Here, the useful anomalous correlation is defined as the correlation of anomalous differences with ideal anomalous differences from the anomalous substructure. The useful anomalous correlation reflects the accuracy of the data and the absence of minor sites. The useful anomalous correlation also reflects the information available for estimating crystallographic phases once the substructure has been determined. In contrast, the anomalous signal (the peak height in a model-phased anomalous difference Fourier at the coordinates of atoms in the anomalous substructure) reflects the information available about each site in the substructure and is related to the ability to find the substructure. A theoretical analysis shows that the expected value of the anomalous signal is the product of the useful anomalous correlation, the square root of the ratio of the number of unique reflections in the data set to the number of sites in the substructure, and a function that decreases with increasing values of the atomic displacement factor for the atoms in the substructure. This means that the ability to find the substructure in a SAD experiment is increased by high data quality and by a high ratio of reflections to sites in the substructure, and is decreased by high atomic displacement factors for the substructure.

A key challenge in the SAD phasing method is solving a structure when the anomalous signal-to-noise ratio is low. Here, algorithms and tools for evaluating and optimizing the useful anomalous correlation and the anomalous signal in a SAD experiment are described. A simple theoretical framework [Terwilliger et al. (2016), Acta Cryst. D72, 346-358] is used to develop methods for planning a SAD experiment, scaling SAD data sets and estimating the useful anomalous correlation and anomalous signal in a SAD data set. The phenix.plan_sad_experiment tool uses a database of solved and unsolved SAD data sets and the expected characteristics of a SAD data set to estimate the probability that the anomalous substructure will be found in the SAD experiment and the expected map quality that would be obtained if the substructure were found. The phenix.scale_and_merge tool scales unmerged SAD data from one or more crystals using local scaling and optimizes the anomalous signal by identifying the systematic differences among data sets, and the phenix.anomalous_signal tool estimates the useful anomalous correlation and anomalous signal after collecting SAD data and estimates the probability that the data set can be solved and the likely figure of merit of phasing.