Integrative Molecular Structure Elucidation and Construction of an Extended Metabolic Pathway Associated with an Ancient Innate Immune Response in COVID-19 Patients.

We present compelling evidence for the existence of an extended innate viperin-dependent pathway, which provides crucial evidence for an adaptive response to viral agents, such as SARS-CoV-2. We show the in vivo biosynthesis of a family of novel endogenous cytosine metabolites with potential antiviral activities. Two-dimensional nuclear magnetic resonance (NMR) spectroscopy revealed a characteristic spin-system motif, indicating the presence of an extended panel of urinary metabolites during the acute viral replication phase. Mass spectrometry additionally enabled the characterization and quantification of the most abundant serum metabolites, showing the potential diagnostic value of the compounds for viral infections. In total, we unveiled ten nucleoside (cytosine- and uracil-based) analogue structures, eight of which were previously unknown in humans allowing us to propose a new extended viperin pathway for the innate production of antiviral compounds. The molecular structures of the nucleoside analogues and their correlation with an array of serum cytokines, including IFN-α2, IFN-γ, and IL-10, suggest an association with the viperin enzyme contributing to an ancient endogenous innate immune defense mechanism against viral infection.

Discovery of a Biotin Synthase That Utilizes an Auxiliary 4Fe-5S Cluster for Sulfur Insertion.

Biotin synthase (BioB) is a member of the Radical SAM superfamily of enzymes that catalyzes the terminal step of biotin (vitamin B7) biosynthesis, in which it inserts a sulfur atom in desthiobiotin to form a thiolane ring. How BioB accomplishes this difficult reaction has been the subject of much controversy, mainly around the source of the sulfur atom. However, it is now widely accepted that the sulfur atom inserted to form biotin stems from the sacrifice of the auxiliary 2Fe-2S cluster of BioB. Here, we bioinformatically explore the diversity of BioBs available in sequence databases and find an unexpected variation in the coordination of the auxiliary iron-sulfur cluster. After in vitro characterization, including the determination of biotin formation and representative crystal structures, we report a new type of BioB utilized by virtually all obligate anaerobic organisms. Instead of a 2Fe-2S cluster, this novel type of BioB utilizes an auxiliary 4Fe-5S cluster. Interestingly, this auxiliary 4Fe-5S cluster contains a ligated sulfide that we propose is used for biotin formation. We have termed this novel type of BioB, Type II BioB, with the E. coli 2Fe-2S cluster sacrificial BioB representing Type I. This surprisingly ubiquitous Type II BioB has implications for our understanding of the function and evolution of Fe-S clusters in enzyme catalysis, highlighting the difference in strategies between the anaerobic and aerobic world.

Structural, Biochemical, and Bioinformatic Basis for Identifying Radical SAM Cyclopropyl Synthases.

The importance of radical S-adenosyl-l-methionine (RS) enzymes in the maturation of ribosomally synthesized and post-translationally modified peptides (RiPPs) continues to expand, specifically for the RS-SPASM subfamily. We recently discovered an RS-SPASM enzyme that installs a carbon-carbon bond between the geminal methyls of valine residues, resulting in the formation of cyclopropylglycine (CPG). Here, we sought to define the family of cyclopropyl (CP) synthases because of the importance of cyclopropane scaffolds in pharmaceutical development. Using RadicalSAM.org, we bioinformatically expanded the family of CP synthases and assigned unique peptide sequences to each subclade. We identified a unique RiPP biosynthetic pathway that encodes a precursor peptide, TigB, with a repeating TIGSVS motif. Using LCMS and NMR techniques, we show that the RS enzyme associated with the pathway, TigE, catalyzes the formation of a methyl-CPG from the conserved isoleucine residing in the repeating motif of TigB. Furthermore, we obtained a crystal structure of TigE, which reveals an unusual tyrosyl ligation to the auxiliary I [4Fe-4S] cluster, provided by a glycine-tyrosine-tryptophan motif unique to all CP synthases. Further, we show that this unique tyrosyl ligation is absolutely required for TigE activity. Together, our results provide insight into how CP synthases perform this unique reaction.

The new epoch of structural insights into radical SAM enzymology.

The Radical SAM (RS) superfamily of enzymes catalyzes a wide array of enzymatic reactions. The majority of these enzymes employ an electron from a reduced [4Fe-4S]+1 cluster to facilitate the reductive cleavage of S-adenosyl-l-methionine, thereby producing a highly reactive 5'-deoxyadenosyl radical (5'-dA⋅) and l-methionine. Typically, RS enzymes use this 5'-dA⋅ to extract a hydrogen atom from the target substrate, starting the cascade of an expansive and impressive variety of chemical transformations. While a great deal of understanding has been gleaned for 5'-dA⋅ formation, because of the chemical diversity within this superfamily, the subsequent chemical transformations have only been fully elucidated in a few examples. In addition, with the advent of new sequencing technology, the size of this family now surpasses 700,000 members, with the number of uncharacterized enzymes and domains also rapidly expanding. In this review, we outline the history of RS enzyme characterization in what we term "epochs" based on advances in technology designed for stably producing these enzymes in an active state. We propose that the state of the field has entered the fourth epoch, which we argue should commence with a protein structure initiative focused solely on RS enzymes to properly tackle this unique superfamily and uncover more novel chemical transformations that likely exist.

Chemoenzymatic Synthesis of 3'-Deoxy-3',4'-didehydro-cytidine triphosphate (ddhCTP).

3'-Deoxy-3',4'-didehydro-cytidine triphosphate (ddhCTP) is a novel antiviral molecule produced by the enzyme viperin during the early stages of the innate immune response. ddhCTP has been shown to act as a chain terminator of flavivirus RNA-dependent RNA polymerases. To date, synthesis of ddhCTP requires complicated synthetic protocols or isolation of the enzyme viperin to catalyze the production of ddhCTP from CTP. Recombinant viperin approaches preclude the production of highly pure ddhCTP (free of contaminants such as CTP), whereas the chemical synthesis involves techniques or equipment not readily available to most laboratories. Herein, we describe the chemoenzymatic synthesis of ddhCTP, starting from commercially available ddhC. We utilize these methods to produce milligram quantities of ddhCTP, ddhCDP, and ddhCMP. Using purified semisynthetic ddhCTP and fully synthetic ddhCTP, we also show ddhCTP does not inhibit NAD+-dependent enzymes such as glyceraldehyde 3-phosphate dehydrogenase, malate dehydrogenase, or lactate dehydrogenase, contrary to a recent report.

CMPK2 restricts Zika virus replication by inhibiting viral translation.

Flaviviruses continue to emerge as global health threats. There are currently no Food and Drug Administration (FDA) approved antiviral treatments for flaviviral infections. Therefore, there is a pressing need to identify host and viral factors that can be targeted for effective therapeutic intervention. Type I interferon (IFN-I) production in response to microbial products is one of the host's first line of defense against invading pathogens. Cytidine/uridine monophosphate kinase 2 (CMPK2) is a type I interferon-stimulated gene (ISG) that exerts antiviral effects. However, the molecular mechanism by which CMPK2 inhibits viral replication is unclear. Here, we report that CMPK2 expression restricts Zika virus (ZIKV) replication by specifically inhibiting viral translation and that IFN-I- induced CMPK2 contributes significantly to the overall antiviral response against ZIKV. We demonstrate that expression of CMPK2 results in a significant decrease in the replication of other pathogenic flaviviruses including dengue virus (DENV-2), Kunjin virus (KUNV) and yellow fever virus (YFV). Importantly, we determine that the N-terminal domain (NTD) of CMPK2, which lacks kinase activity, is sufficient to restrict viral translation. Thus, its kinase function is not required for CMPK2's antiviral activity. Furthermore, we identify seven conserved cysteine residues within the NTD as critical for CMPK2 antiviral activity. Thus, these residues may form an unknown functional site in the NTD of CMPK2 contributing to its antiviral function. Finally, we show that mitochondrial localization of CMPK2 is required for its antiviral effects. Given its broad antiviral activity against flaviviruses, CMPK2 is a promising potential pan-flavivirus inhibitor.

Reductions in regional theta power and fronto-parietal theta-gamma phase-amplitude coupling during gaze processing in bipolar disorder.

Impaired social cognition is common in bipolar disorder (BD) and predicts poor functional outcomes. A critical determinant of social cognition is the ability to discriminate others' gaze direction, and its alteration may contribute to functional impairment in BD. However, the neural mechanisms underlying gaze processing in BD are unclear. Because neural oscillations are crucial neurobiological mechanisms supporting cognition, we aimed to understand their role in gaze processing in BD. Using electroencephalography (EEG) data recorded during a gaze discrimination task for 38 BD and 34 controls (HC), we examined: theta and gamma power over bilateral posterior and midline anterior locations associated with early face processing and higher-level cognitive processing, and theta-gamma phase-amplitude coupling (PAC) between locations. Compared to HC, BD showed reduced midline-anterior and left-posterior theta power, and diminished bottom-up/top-down theta-gamma PAC between anterior/posterior sites. Reduced theta power and theta-gamma PAC related to slower response times. These findings suggest that altered theta oscillations and anterior-posterior cross-frequency coupling between areas associated with higher-level cognition and early face processing may underlie impaired gaze processing in BD. This is a crucial step towards translational research that may inform novel social cognitive interventions (e.g., neuromodulation to target specific oscillatory dynamics) to improve functioning in BD.

Abnormally Enhanced Midfrontal Theta Activity During Response Monitoring in Youths With Obsessive-Compulsive Disorder.

BACKGROUND: Response monitoring, as reflected in electroencephalogram recordings after commission of errors, has been consistently shown to be abnormally enhanced in individuals with obsessive-compulsive disorder (OCD). This has traditionally been quantified as error-related negativity (ERN) and may reflect abnormal neurophysiological mechanisms underlying OCD. However, the ERN reflects the increase in phase-locked activities, particularly in the theta-band (4-8 Hz), and does not reflect non-phase-locked activities. To more broadly investigate midfrontal theta activity in a brain region that is essential for complex cognition, this study investigated theta abnormalities during response monitoring in participants with OCD to acheive a better understanding of the mechanism underlying the ERN. METHODS: Electroencephalogram data were recorded from 99 participants with pediatric OCD and 99 sex- and age-matched healthy control participants while they completed the arrow flanker task. Effects of group (OCD, healthy control) and response type (error, correct) on postresponse theta total power and intertrial phase coherence (ITPC) were examined using mixed analysis of covariance and Bayesian analyses controlling for sex and accuracy. RESULTS: Theta total power was larger on error than on correct trials and larger in OCD than healthy control participants, but there was no effect of response type between groups. Theta ITPC was larger on error than correct trials, but there was no group difference or response type difference between the groups. Correlations of theta total power and ITPC with clinical measures were overall small. CONCLUSIONS: Abnormally enhanced midfrontal theta total power, but not ITPC, may reflect ineffective heightened response monitoring or compensatory activity in pediatric OCD.

Reduced theta-band neural oscillatory activity during affective cognitive control in bipolar I disorder.

Individuals with bipolar I disorder (BD) have difficulty inhibiting context-inappropriate responses. However, neural mechanisms of impaired cognitive control over impulsive behaviors, especially in response to emotion, are unclear. Theta-band neural oscillatory activity over midfrontal areas is thought to reflect cognitive control. The current study examined behavioral performance and theta-band activity during inhibition to affective stimuli in BD, relative to healthy control participants (HC). Sixty-seven participants with BD and 48 HC completed a Go/No-Go task with emotional face stimuli during electroencephalography (EEG) recording. Behavior was measured with reaction time, discriminability (d') and response bias (ß). Time-frequency decomposition of EEG data was used to extract event-related theta-band (4-7 Hz) neural oscillatory power and inter-trial phase consistency (ITPC) over midline fronto-central areas. Behavior and theta-band activity were compared between groups, while covarying for age. Participants with BD exhibited slower response execution times on correct Go trials and reduced behavioral discrimination of emotional versus neutral faces, compared to HC. Theta-band power and ITPC were reduced in BD relative to HC. Theta-band power was higher on No-Go trials than Go trials. The magnitude of differences in theta-band activity between Go/No-Go trial types did not differ between groups. Increased theta-band power was associated with faster response execution times, greater discrimination of differing facial expressions, and stronger tendency to respond both across the full sample and within the BD group. Attenuated midline fronto-central theta-band activity may contribute to reduced cognitive control and maladaptive behavioral responding to emotional cues in individuals with BD.

Event-related potential correlates of affective response inhibition in bipolar I disorder: Comparison with schizophrenia.

BACKGROUND: Individuals with bipolar I disorder (BD) have difficulty inhibiting context-inappropriate responses. The neural mechanisms contributing to these difficulties, especially in emotional contexts, are little understood. This study aimed to inform mechanisms of impaired impulsivity control in response to emotion in BD, and whether response inhibition indices are altered to a similar degree in schizophrenia spectrum disorders (SZ). We examined alterations to behavioral performance and event-related potentials (ERPs) during inhibition to affective stimuli in BD, relative to healthy control participants (HC) and SZ. METHODS: Sixty-six participants with BD, 32 participants with SZ, and 48 HC completed a Go/No-Go task with emotional face stimuli while electroencephalography was recorded. Behavioral signal detection metrics (perceptual sensitivity, response bias) and ERPs (N200, P300) were compared across groups. RESULTS: Relative to HC, participants with BD showed reduced (1) discrimination of Go vs. No-Go stimuli (i.e., emotional vs. neutral faces), and (2) P300 amplitudes elicited by emotional faces. Results similarly extended to SZ: BD and SZ groups did not differ on behavioral performance nor ERP amplitudes. LIMITATIONS: Aspects of the Go/No-Go task design may have limited findings, and medication effects on ERP amplitudes in patient samples cannot be fully ruled out. CONCLUSIONS: Findings suggest the difficulty participants with BD and SZ experienced on the current affective response inhibition task lied largely in discriminating between facial expressions. Difficulties with discriminating emotional from neutral expressions may contribute to difficulties with appropriate behavioral responding in social-affective contexts for individuals with BD and SZ.

Viperin triggers ribosome collision-dependent translation inhibition to restrict viral replication.

Innate immune responses induce hundreds of interferon-stimulated genes (ISGs). Viperin, a member of the radical S-adenosyl methionine (SAM) superfamily of enzymes, is the product of one such ISG that restricts the replication of a broad spectrum of viruses. Here, we report a previously unknown antiviral mechanism in which viperin activates a ribosome collision-dependent pathway that inhibits both cellular and viral RNA translation. We found that the radical SAM activity of viperin is required for translation inhibition and that this is mediated by viperin's enzymatic product, 3'-deoxy-3',4'-didehydro-CTP (ddhCTP). Viperin triggers ribosome collisions and activates the MAPKKK ZAK pathway that in turn activates the GCN2 arm of the integrated stress response pathway to inhibit translation. The study illustrates the importance of translational repression in the antiviral response and identifies viperin as a translation regulator in innate immunity.

Affective Dysregulation Precedes Emergence of Psychosis-Like Experiences in a Community Sample of Young Adults.

Affective dysregulation (AD) among persons with schizophrenia spectrum disorders, involving the tendency to exhibit sensitivity to minor stress and negative affective states, is an important diagnostic feature and relates to poorer functional and clinical outcomes. Studies of persons with elevated risk for psychosis demonstrate similar AD to those with schizophrenia, and literature suggest a potential influence of AD in the transition from psychosis-like symptoms (PLEs) to disorder. Cross-sectional investigations to date have supported the link between AD and psychosis, and longitudinal studies have mostly yielded mixed findings without demonstration of potential causal relationships between AD and psychosis. This study examined the concurrent and predictive relationships between AD and PLE in a community sample of youth (n = 630) with attention to distinct facets of AD as a latent construct, including low resiliency, low reactive control, and negative emotionality, using structural equation to estimate a longitudinal cross-lagged and autoregressive model across 3 study waves from 15 to 24 years of age. As hypothesized, AD in the mid-teen years predicted subsequent PLE 3 years later. In addition, we found that increasing PLE in the end of the teen years related to a subsequent increase in AD in the early 20s. A cross-sectional relationship between AD and PLE in the mid-teen years was also supported. Findings overall describe important relationships between AD and PLE that appear to vary with developmental stage, implicating various factors to inform approaches for identifying youth who may be at risk for subsequent PLE or other mental health conditions.

Inhibition of SARS-CoV-2 polymerase by nucleotide analogs from a single-molecule perspective.

The absence of 'shovel-ready' anti-coronavirus drugs during vaccine development has exceedingly worsened the SARS-CoV-2 pandemic. Furthermore, new vaccine-resistant variants and coronavirus outbreaks may occur in the near future, and we must be ready to face this possibility. However, efficient antiviral drugs are still lacking to this day, due to our poor understanding of the mode of incorporation and mechanism of action of nucleotides analogs that target the coronavirus polymerase to impair its essential activity. Here, we characterize the impact of remdesivir (RDV, the only FDA-approved anti-coronavirus drug) and other nucleotide analogs (NAs) on RNA synthesis by the coronavirus polymerase using a high-throughput, single-molecule, magnetic-tweezers platform. We reveal that the location of the modification in the ribose or in the base dictates the catalytic pathway(s) used for its incorporation. We show that RDV incorporation does not terminate viral RNA synthesis, but leads the polymerase into backtrack as far as 30 nt, which may appear as termination in traditional ensemble assays. SARS-CoV-2 is able to evade the endogenously synthesized product of the viperin antiviral protein, ddhCTP, though the polymerase incorporates this NA well. This experimental paradigm is essential to the discovery and development of therapeutics targeting viral polymerases.

To multiply and spread from cell to cell, the virus responsible for COVID-19 (also known as SARS-CoV-2) must first replicate its genetic information. This process involves a 'polymerase' protein complex making a faithful copy by assembling a precise sequence of building blocks, or nucleotides. The only drug approved against SARS-CoV-2 by the US Food and Drug Administration (FDA), remdesivir, consists of a nucleotide analog, a molecule whose structure is similar to the actual building blocks needed for replication. If the polymerase recognizes and integrates these analogs into the growing genetic sequence, the replication mechanism is disrupted, and the virus cannot multiply. Most approaches to study this process seem to indicate that remdesivir works by stopping the polymerase and terminating replication altogether. Yet, exactly how remdesivir and other analogs impair the synthesis of new copies of the virus remains uncertain. To explore this question, Seifert, Bera et al. employed an approach called magnetic tweezers which uses a magnetic field to manipulate micro-particles with great precision. Unlike other methods, this technique allows analogs to be integrated under conditions similar to those found in cells, and to be examined at the level of a single molecule. The results show that contrary to previous assumptions, remdesivir does not terminate replication; instead, it causes the polymerase to pause and backtrack (which may appear as termination in other techniques). The same approach was then applied to other nucleotide analogs, some of which were also found to target the SARS-CoV-2 polymerase. However, these analogs are incorporated differently to remdesivir and with less efficiency. They also obstruct the polymerase in distinct ways. Taken together, the results by Seifert, Bera et al. suggest that magnetic tweezers can be a powerful approach to reveal how analogs interfere with replication. This information could be used to improve currently available analogs as well as develop new antiviral drugs that are more effective against SARS-CoV-2. This knowledge will be key at a time when treatments against COVID-19 are still lacking, and may be needed to protect against new variants and future outbreaks.

Structural basis for tRNA methylthiolation by the radical SAM enzyme MiaB.

Numerous post-transcriptional modifications of transfer RNAs have vital roles in translation. The 2-methylthio-N6-isopentenyladenosine (ms2i6A) modification occurs at position 37 (A37) in transfer RNAs that contain adenine in position 36 of the anticodon, and serves to promote efficient A:U codon-anticodon base-pairing and to prevent unintended base pairing by near cognates, thus enhancing translational fidelity1-4. The ms2i6A modification is installed onto isopentenyladenosine (i6A) by MiaB, a radical S-adenosylmethionine (SAM) methylthiotransferase. As a radical SAM protein, MiaB contains one [Fe4S4]RS cluster used in the reductive cleavage of SAM to form a 5'-deoxyadenosyl 5'-radical, which is responsible for removing the C2 hydrogen of the substrate5. MiaB also contains an auxiliary [Fe4S4]aux cluster, which has been implicated6-9 in sulfur transfer to C2 of i6A37. How this transfer takes place is largely unknown. Here we present several structures of MiaB from Bacteroides uniformis. These structures are consistent with a two-step mechanism, in which one molecule of SAM is first used to methylate a bridging µ-sulfido ion of the auxiliary cluster. In the second step, a second SAM molecule is cleaved to a 5'-deoxyadenosyl 5'-radical, which abstracts the C2 hydrogen of the substrate but only after C2 has undergone rehybridization from sp2 to sp3. This work advances our understanding of how enzymes functionalize inert C-H bonds with sulfur.

Chemical Synthesis of the Antiviral Nucleotide Analogue ddhCTP.

3'-Deoxy-3',4'-didehydro-cytidine triphosphate (ddhCTP) is a novel antiviral molecule produced by the enzyme viperin as part of the innate immune response. ddhCTP has been shown to act as an obligate chain terminator of flavivirus and SARS-CoV-2 RNA-dependent RNA polymerases; however, further biophysical studies have been precluded by limited access to this promising antiviral. Herein, we report a robust and scalable synthesis of ddhCTP as well as the mono- and diphosphates ddhCMP and ddhCDP, respectively. Identification of a 2'-silyl ether protection strategy enabled selective synthesis and facile purification of the 5'-triphosphate, culminating in the preparation of ddhCTP on a gram scale.

Structural Insight into the Substrate Scope of Viperin and Viperin-like Enzymes from Three Domains of Life.

Viperin is a member of the radical S-adenosylmethionine superfamily and has been shown to restrict the replication of a wide range of RNA and DNA viruses. We recently demonstrated that human viperin (HsVip) catalyzes the conversion of CTP to 3'-deoxy-3',4'-didehydro-CTP (ddhCTP or ddh-synthase), which acts as a chain terminator for virally encoded RNA-dependent RNA polymerases from several flaviviruses. Viperin homologues also exist in non-chordate eukaryotes (e.g., Cnidaria and Mollusca), numerous fungi, and members of the archaeal and eubacterial domains. Recently, it was reported that non-chordate and non-eukaryotic viperin-like homologues are also ddh-synthases and generate a diverse range of ddhNTPs, including the newly discovered ddhUTP and ddhGTP. Herein, we expand on the catalytic mechanism of mammalian, fungal, bacterial, and archaeal viperin-like enzymes with a combination of X-ray crystallography and enzymology. We demonstrate that, like mammalian viperins, these recently discovered viperin-like enzymes operate through the same mechanism and can be classified as ddh-synthases. Furthermore, we define the unique chemical and physical determinants supporting ddh-synthase activity and nucleotide selectivity, including the crystallographic characterization of a fungal viperin-like enzyme that utilizes UTP as a substrate and a cnidaria viperin-like enzyme that utilizes CTP as a substrate. Together, these results support the evolutionary conservation of the ddh-synthase activity and its broad phylogenetic role in innate antiviral immunity.

Inhibition of SARS-CoV-2 polymerase by nucleotide analogs: a single molecule perspective.

The nucleotide analog Remdesivir (RDV) is the only FDA-approved antiviral therapy to treat infection by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The physical basis for efficient utilization of RDV by SARS-CoV-2 polymerase is unknown. Here, we characterize the impact of RDV and other nucleotide analogs on RNA synthesis by the polymerase using a high-throughput, single-molecule, magnetic-tweezers platform. The location of the modification in the ribose or in the base dictates the catalytic pathway(s) used for its incorporation. We reveal that RDV incorporation does not terminate viral RNA synthesis, but leads the polymerase into deep backtrack, which may appear as termination in traditional ensemble assays. SARS-CoV-2 is able to evade the endogenously synthesized product of the viperin antiviral protein, ddhCTP, though the polymerase incorporates this nucleotide analog well. This experimental paradigm is essential to the discovery and development of therapeutics targeting viral polymerases. TEASER: We revise Remdesivir's mechanism of action and reveal SARS-CoV-2 ability to evade interferon-induced antiviral ddhCTP.

Structural basis for non-radical catalysis by TsrM, a radical SAM methylase.

Tryptophan 2C methyltransferase (TsrM) methylates C2 of the indole ring of L-tryptophan during biosynthesis of the quinaldic acid moiety of thiostrepton. TsrM is annotated as a cobalamin-dependent radical S-adenosylmethionine (SAM) methylase; however, TsrM does not reductively cleave SAM to the universal 5'-deoxyadenosyl 5'-radical intermediate, a hallmark of radical SAM (RS) enzymes. Herein, we report structures of TsrM from Kitasatospora setae, which are the first structures of a cobalamin-dependent radical SAM methylase. Unexpectedly, the structures show an essential arginine residue that resides in the proximal coordination sphere of the cobalamin cofactor, and a [4Fe-4S] cluster that is ligated by a glutamyl residue and three cysteines in a canonical CXXXCXXC RS motif. Structures in the presence of substrates suggest a substrate-assisted mechanism of catalysis, wherein the carboxylate group of SAM serves as a general base to deprotonate N1 of the tryptophan substrate, facilitating the formation of a C2 carbanion.