Tubulin code eraser CCP5 binds branch glutamates by substrate deformation.

Microtubule function is modulated by the tubulin code, diverse posttranslational modifications that are altered dynamically by writer and eraser enzymes1. Glutamylation-the addition of branched (isopeptide-linked) glutamate chains-is the most evolutionarily widespread tubulin modification2. It is introduced by tubulin tyrosine ligase-like enzymes and erased by carboxypeptidases of the cytosolic carboxypeptidase (CCP) family1. Glutamylation homeostasis, achieved through the balance of writers and erasers, is critical for normal cell function3-9, and mutations in CCPs lead to human disease10-13. Here we report cryo-electron microscopy structures of the glutamylation eraser CCP5 in complex with the microtubule, and X-ray structures in complex with transition-state analogues. Combined with NMR analysis, these analyses show that CCP5 deforms the tubulin main chain into a unique turn that enables lock-and-key recognition of the branch glutamate in a cationic pocket that is unique to CCP family proteins. CCP5 binding of the sequences flanking the branch point primarily through peptide backbone atoms enables processing of diverse tubulin isotypes and non-tubulin substrates. Unexpectedly, CCP5 exhibits inefficient processing of an abundant ß-tubulin isotype in the brain. This work provides an atomistic view into glutamate branch recognition and resolution, and sheds light on homeostasis of the tubulin glutamylation syntax.

Anti-CGRP antibody galcanezumab modifies the function of the trigeminovascular nocisensor complex in the rat.

BACKGROUND: Monoclonal antibodies directed against the neuropeptide calcitonin gene-related peptide (CGRP) are effective in the prevention of chronic and frequent episodic migraine. Since the antibodies do not cross the blood brain barrier, their antinociceptive effect is attributed to effects in meningeal tissues. We aimed to probe if such an antibody can be visualized within the dura mater and the trigeminal ganglia following its administration to rats and to examine if the activity of the trigeminovascular nocisensor complex is influenced by this treatment. METHODS: Effects of the anti-CGRP antibody galcanezumab on the trigeminovascular nocisensor complex was examined by measuring release of sensory neuropeptides and histamine from the rat dura mater. Deposits of galcanezumab were visualized by fluorescence microscopy in the trigeminal ganglion and the dura mater. RESULTS: Fluorophore-labelled galcanezumab was detected in the dura mater and the trigeminal ganglion up to 30 days after treatment affirming the long-lasting modulatory effect of this antibody. In female rats, seven days after systemic treatment with galcanezumab the capsaicin-induced release of CGRP was decreased, while that of substance P (SP) was increased in the dura mater. In control rats, release of the inhibitory neuropeptide somatostatin (SOM) was higher in females than in males. Stimulation with high concentration of KCl did not significantly change the release of SOM in control animals, while in rats treated with galcanezumab SOM release was slightly reduced. Galcanezumab treatment also reduced the amount of histamine released from dural mast cells upon stimulation with CGRP, while the effect of compound 48/80 on histamine release was not changed. CONCLUSIONS: Galcanezumab treatment is followed by multiple changes in the release of neuropeptides and histamine in the trigeminal nocisensor complex, which may contribute to the migraine preventing effect of anti-CGRP antibodies. These changes affecting the communication between the components of the trigeminal nocisensor complex may reduce pain susceptibility in migraine patients treated with CGRP targeting monoclonal antibodies.

Aldehyde-Based Inhibitors of the Peptidoglycan O-Acetylesterase Ape.

The O-acetylation of the muramic acid residues in peptidoglycan (PG) is a modification that protects the bacteria from lysis due to the action of lysozyme. In Gram-negative bacteria, deacetylation is required to allow lytic transglycosylases to promote PG cleavage during cell growth and division. This deacetylation is catalyzed by O-acetylpeptidoglycan esterase (Ape) which is a serine esterase and employs covalent catalysis via a serine-linked acyl enzyme intermediate. Loss of Ape activity affects the size and shape of bacteria and dramatically reduces virulence. In this work, we report the first rationally designed aldehyde-based inhibitors of Ape from Campylobacter jejuni. The most potent of these acts as a competitive inhibitor with a Ki value of 13 µM. We suspect that the inhibitors are forming adducts with the active site serine that closely mimic the tetrahedral intermediate of the normal catalytic cycle. Support for this notion is found in the observation that reduction of the aldehyde to an alcohol effectively abolishes the inhibition.

Peptidoglycan binding by a pocket on the accessory NTF2-domain of Pgp2 directs helical cell shape of Campylobacter jejuni.

The helical morphology of Campylobacter jejuni, a bacterium involved in host gut colonization and pathogenesis in humans, is determined by the structure of the peptidoglycan (PG) layer. This structure is dictated by trimming of peptide stems by the LD-carboxypeptidase Pgp2 within the periplasm. The interaction interface between Pgp2 and PG to select sites for peptide trimming is unknown. We determined a 1.6 Å resolution crystal structure of Pgp2, which contains a conserved LD-carboxypeptidase domain and a previously uncharacterized domain with an NTF2-like fold (NTF2). We identified a pocket in the NTF2 domain formed by conserved residues and located ∼40 Å from the LD-carboxypeptidase active site. Expression of pgp2 in trans with substitutions of charged (Lys257, Lys307, Glu324) and hydrophobic residues (Phe242 and Tyr233) within the pocket did not restore helical morphology to a pgp2 deletion strain. Muropeptide analysis indicated a decrease of murotripeptides in the deletion strain expressing these mutants, suggesting reduced Pgp2 catalytic activity. Pgp2 but not the K307A mutant was pulled down by C. jejuni Δpgp2 PG sacculi, supporting a role for the pocket in PG binding. NMR spectroscopy was used to define the interaction interfaces of Pgp2 with several PG fragments, which bound to the active site within the LD-carboxypeptidase domain and the pocket of the NTF2 domain. We propose a model for Pgp2 binding to PG strands involving both the LD-carboxypeptidase domain and the accessory NTF2 domain to induce a helical cell shape.

Recent Advances in the Synthesis and Biological Applications of Peptidoglycan Fragments.

The biosynthesis, breakdown, and modification of peptidoglycan (PG) play vital roles in both bacterial viability and in the response of human physiology to bacterial infection. Studies on PG biochemistry are hampered by the fact that PG is an inhomogeneous insoluble macromolecule. Chemical synthesis is therefore an important means to obtain PG fragments that may serve as enzyme substrates and elicitors of the human immune response. This review outlines the recent advances in the synthesis and biochemical studies of PG fragments, PG biosynthetic intermediates (such as Park's nucleotides and PG lipids), and PG breakdown products (such as muramyl dipeptides and anhydro-muramic acid-containing fragments). A rich variety of synthetic approaches has been applied to preparing such compounds since carbohydrate, peptide, and phospholipid chemical methodologies must all be applied.

A guanidinium group is an effective mimic of the tertiary carbocation formed by isopentenyl diphosphate isomerase.

Type I isopentenyl diphosphate isomerase is a metal-dependent enzyme that generates a tertiary carbocation intermediate during catalysis. This study describes an inhibitor (2-guanidinoethyl(dihydroxyphosphorylmethyl)phosphinate) of the isomerase that bears a guanidinium as a carbocation mimic and a phosphinylphosphonate as a non-hydrolyzable metal binding functionality. Inhibition kinetics show that the compound acts in a competitive manner with a Ki value of 129 nM (KM,IPP/Ki = 27). An analogous inhibitor bearing a tertiary ammonium as the carbocation mimic was 50-fold less potent, suggesting that the planar guanidinium is a more effective carbocation mimic. Inhibitors bearing an acylated methanesulfonamide or a hydroxamate group in place of the pyrophosphate inhibited the enzyme at millimolar concentrations indicating that the isomerase is highly specific for binding to the diphosphate portion of the intermediate.

Forecasting for COVID-19 has failed.

Epidemic forecasting has a dubious track-record, and its failures became more prominent with COVID-19. Poor data input, wrong modeling assumptions, high sensitivity of estimates, lack of incorporation of epidemiological features, poor past evidence on effects of available interventions, lack of transparency, errors, lack of determinacy, consideration of only one or a few dimensions of the problem at hand, lack of expertise in crucial disciplines, groupthink and bandwagon effects, and selective reporting are some of the causes of these failures. Nevertheless, epidemic forecasting is unlikely to be abandoned. Some (but not all) of these problems can be fixed. Careful modeling of predictive distributions rather than focusing on point estimates, considering multiple dimensions of impact, and continuously reappraising models based on their validated performance may help. If extreme values are considered, extremes should be considered for the consequences of multiple dimensions of impact so as to continuously calibrate predictive insights and decision-making. When major decisions (e.g. draconian lockdowns) are based on forecasts, the harms (in terms of health, economy, and society at large) and the asymmetry of risks need to be approached in a holistic fashion, considering the totality of the evidence.

A guanidinium-based inhibitor of a type I isopentenyl diphosphate isomerase.

An inhibitor bearing a phosphinylphosphonate group appended to a guanidinium functionality was designed to inhibit enzymes that generate carbocations from dimethylallyl diphosphate. When tested against human farnesyl diphosphate synthase the inhibitor bound with high micromolar affinity and did not bind more tightly than an isosteric inhibitor lacking the guanidinium functionality. When tested against the Type I isopentenyl diphosphate:dimethylallyl diphosphate isomerase from Escherichia coli, the inhibitor bound with a Ki value of 120 nM, which was 400 times greater than its isosteric counterpart. This strategy of inhibition was much more effective with an enzyme that generates a carbocation that is not stabilized by both resonance and ion pairing, presumably because there is more evolutionary pressure on the enzyme to stabilize the cation.

A case study in model failure? COVID-19 daily deaths and ICU bed utilisation predictions in New York state.

Forecasting models have been influential in shaping decision-making in the COVID-19 pandemic. However, there is concern that their predictions may have been misleading. Here, we dissect the predictions made by four models for the daily COVID-19 death counts between March 25 and June 5 in New York state, as well as the predictions of ICU bed utilisation made by the influential IHME model. We evaluated the accuracy of the point estimates and the accuracy of the uncertainty estimates of the model predictions. First, we compared the "ground truth" data sources on daily deaths against which these models were trained. Three different data sources were used by these models, and these had substantial differences in recorded daily death counts. Two additional data sources that we examined also provided different death counts per day. For accuracy of prediction, all models fared very poorly. Only 10.2% of the predictions fell within 10% of their training ground truth, irrespective of distance into the future. For accurate assessment of uncertainty, only one model matched relatively well the nominal 95% coverage, but that model did not start predictions until April 16, thus had no impact on early, major decisions. For ICU bed utilisation, the IHME model was highly inaccurate; the point estimates only started to match ground truth after the pandemic wave had started to wane. We conclude that trustworthy models require trustworthy input data to be trained upon. Moreover, models need to be subjected to prespecified real time performance tests, before their results are provided to policy makers and public health officials.

Peptides Containing meso-Oxa-Diaminopimelic Acid as Substrates for the Cell-Shape-Determining Proteases Csd6 and Pgp2.

The enzymes Csd6 and Pgp2 are peptidoglycan (PG) proteases found in the pathogenic bacteria Helicobacter pylori and Campylobacter jejuni, respectively. These enzymes are involved in the trimming of non-crosslinked PG sidechains and catalyze the cleavage of the bond between meso-diaminopimelic acid (meso-Dap) and d-alanine, thus converting a PG tetrapeptide into a PG tripeptide. They are known to be cell-shape-determining enzymes, because deletion of the corresponding genes results in mutant strains that have lost the normal helical phenotype and instead possess a straight-rod morphology. In this work, we report two approaches directed towards the synthesis of the tripeptide substrate Ac-iso-d-Glu-meso-oxa-Dap-d-Ala, which serves as a mimic of the terminus of an non-crosslinked PG tetrapeptide substrate. The isosteric analogue meso-oxa-Dap was utilized in place of meso-Dap to simplify the synthetic procedure. The more efficient synthesis involved ring opening of a peptide-embedded aziridine by a serine-based nucleophile. A branched tetrapeptide was also prepared as a mimic of the terminus of a crosslinked PG tetrapeptide. We used MS analysis to demonstrate that the tripeptide serves as a substrate for both Csd6 and Pgp2 and that the branched tetrapeptide serves as a substrate for Pgp2, albeit at a significantly slower rate.

Studies with Guanidinium- and Amidinium-Based Inhibitors Suggest Minimal Stabilization of Allylic Carbocation Intermediates by Dehydrosqualene and Squalene Synthases.

Dehydrosqualene and squalene synthases catalyze the redox neutral and the reductive, head-to-head dimerization of farnesyl diphosphate, respectively. In each case, the reaction is thought to proceed via an initial dissociation of farnesyl diphosphate to form an allylic carbocation-pyrophosphate ion pair. This work describes the synthesis and testing of inhibitors in which a guanidinium or amidinium moiety is flanked by a phosphonylphosphinate group and a hydrocarbon tail. These functional groups bear a planar, delocalized, positive charge and therefore should act as excellent mimics of an allylic carbocation. An inhibitor bearing a neutral urea moiety was also prepared as a control. The positively charged inhibitors acted as competitive inhibitors against Staphylococcus aureus dehydrosqualene synthase with Ki values in the low micromolar range. Surprisingly, the neutral urea inhibitor was the most potent of the three. Similar trends were seen with the first half reaction of human squalene synthase. One interpretation of these results is that the active sites of these enzymes do not directly stabilize the allylic carbocation via electrostatic or π-cation interactions. Instead, it is likely that the enzymes use tight binding to the pyrophosphate and lipid moieties to promote catalysis and that electrostatic stabilization of the carbocation is provided by the bound pyrophosphate product. An alternate possibility is that these inhibitors cannot bind to the "ionization FPP-binding site" of the enzyme and only bind to the "nonionizing FPP-binding site". In either case, all reported attempts to generate potent inhibitors with cationic FPP analogues have been unsuccessful to date.

Replacing the Argon ICP: Nitrogen Microwave Inductively Coupled Atmospheric-Pressure Plasma (MICAP) for Mass Spectrometry.

We combine a recently developed high-power, nitrogen-sustained microwave plasma source-the Microwave Inductively Coupled Atmospheric-Pressure Plasma (MICAP)-with time-of-flight mass spectrometry (TOFMS) and provide the first characterization of this elemental mass spectrometry configuration. Motivations for assessment of this ionization source are scientific and budgetary: unlike the argon-sustained Inductively Coupled Plasma (ICP), the MICAP is sustained with nitrogen, which eliminates high operating costs associated with argon-gas consumption. Additionally, use of a commercial grade magnetron for microwave generation simplifies plasma-powering electronics. In this study, we directly compare MICAP-TOFMS performance with that of an argon-ICP as the atomic ionization source on the same TOFMS instrument. Initial results with the MICAP source demonstrate limits of detection and sensitivities that are, for most elements, on par with those of the ICP-TOFMS. The N2-MICAP source provides a much "cleaner" background spectrum than the ICP; absence of argon-based interferences greatly simplifies analysis of isotopes such as 40Ca, 56Fe, and 75As, which typically suffer from spectral interferences in ICP-MS. The major plasma species measured from the N2-MICAP source include NO+, N2+, N+, N3+, O2+, N4+, and H2O+; we observed no plasma-background species above mass-to-charge 60. Absence of troublesome argon-based spectral interferences is a compelling advantage of the MICAP source. For example, with MICAP-TOFMS, the limit of detection for arsenic is less than 100 ng L-1 even in a 1% NaCl solution; with ICP-MS, 35Cl40Ar+ interferes with 75As+ and arsenic analysis is difficult-to-impossible in chlorine-containing matrices.

Helical shape of Helicobacter pylori requires an atypical glutamine as a zinc ligand in the carboxypeptidase Csd4.

Peptidoglycan modifying carboxypeptidases (CPs) are important determinants of bacterial cell shape. Here, we report crystal structures of Csd4, a three-domain protein from the human gastric pathogen Helicobacter pylori. The catalytic zinc in Csd4 is coordinated by a rare His-Glu-Gln configuration that is conserved among most Csd4 homologs, which form a distinct subfamily of CPs. Substitution of the glutamine to histidine, the residue found in prototypical zinc carboxypeptidases, resulted in decreased enzyme activity and inhibition by phosphate. Expression of the histidine variant at the native locus in a H. pylori csd4 deletion strain did not restore the wild-type helical morphology. Biochemical assays show that Csd4 can cleave a tripeptide peptidoglycan substrate analog to release m-DAP. Structures of Csd4 with this substrate analog or product bound at the active site reveal determinants of peptidoglycan specificity and the mechanism to cleave an isopeptide bond to release m-DAP. Our data suggest that Csd4 is the archetype of a new CP subfamily with a domain scheme that differs from this large family of peptide-cleaving enzymes.

Mechanistic studies on the indole prenyltransferases.

Covering: up to 2014. Prenylated indole alkaloids comprise a large and structurally diverse family of natural products that often display potent biological activities. In recent years a large family of prenyltransferases that install prenyl groups onto the indole core have been discovered. While the vast majority of these enzymes are evolutionarily related and share a common protein fold, they are remarkably versatile in their ability to catalyze reverse and normal prenylations at all positions on the indole ring. This highlight article will focus on recent studies of the mechanisms utilized by indole prenyltransferases. While all of the prenylation reactions may follow a direct electrophilic aromatic substitution mechanism, studies of structure and reactivity suggest that in some cases prenylation may first occur at the nucleophilic C-3 position, and subsequent rearrangements then generate the final product.

A methyltransferase initiates terpene cyclization in teleocidin B biosynthesis.

Teleocidin B is an indole terpenoid isolated from Streptomyces. Due to its unique chemical structure and ability to activate protein kinase C, it has attracted interest in the areas of organic chemistry and cell biology. Here, we report the identification of genes encoding enzymes for teleocidin B biosynthesis, including nonribosomal peptide synthetase (tleA), P-450 monooxygenase (tleB), prenyltransferase (tleC), and methyltransferase (tleD). The tleD gene, which is located outside of the tleABC cluster on the chromosome, was identified by transcriptional analysis and heterologous expression. Remarkably, TleD not only installs a methyl group on the geranyl moiety of the precursor but also facilitates the nucleophilic attack from the electron-rich indole to the resultant cation, to form the indole-fused six-membered ring. This is the first demonstration of a cation, generated from methylation, triggering successive terpenoid ring closure.

Simultaneous mass quantification of nanoparticles of different composition in a mixture by microdroplet generator-ICPTOFMS.

This work investigated the potential of a high temporal resolution inductively coupled plasma time-of-flight mass spectrometer (ICPTOFMS) in combination with a microdroplet generator (MDG) for simultaneous mass quantification of different nanoparticles (NPs) in a mixture. For this purpose, a test system containing certified Au NPs, well characterized Ag NPs, and core-shell NPs composed of an Au core and an Ag shell was employed. Thanks to the full spectra coverage and rapid simultaneous detection of the TOFMS, the element composition of individual particles can be determined. The pure Ag NPs and the core-shell NPs could be differentiated despite the same mass of Ag they contain. Calibration with monodisperse droplets consisting of standard solutions allowed for the mass quantification of NPs without the use of NP certified materials. On the basis of this mass quantification, the sizes of NPs originating from the same aqueous suspension were simultaneously determined with an accuracy of 7-12%. The size-equivalent limits of detection estimated with the 3*σ criterion were 13 nm for Au and 16 nm for Ag. Estimation of the LODs using Poisson statistics resulted in 19 and 27 nm, respectively. In addition, the 30 µs temporal resolution of the ICPTOFMS allowed studying interactions of NPs with the ICP based on their transient MS signals. The results demonstrated a difference in vaporization behavior of the core-shell NPs and solutions and indicated that vaporization of the Ag shell takes place prior to the Au core.

Structure of MurNAc 6-phosphate hydrolase (MurQ) from Haemophilus influenzae with a bound inhibitor.

The breakdown and recycling of peptidoglycan, an essential polymeric cell structure, occur in a number of bacterial species. A key enzyme in the recycling pathway of one of the components of the peptidoglycan layer, N-acetylmuramic acid (MurNAc), is MurNAc 6-phosphate hydrolase (MurQ). This enzyme catalyzes the cofactor-independent cleavage of a relatively nonlabile ether bond and presents an interesting target for mechanistic studies. Open chain product and substrate analogues were synthesized and tested as competitive inhibitors (K(is) values of 1.1 ± 0.3 and 0.23 ± 0.02 mM, respectively) of the MurNAc 6P hydrolase from Escherichia coli (MurQ-EC). To identify the roles of active site residues that are important for catalysis, the substrate analogue was cocrystallized with the MurNAc 6P hydrolase from Haemophilus influenzae (MurQ-HI) that was amenable to crystallographic studies. The cocrystal structure of MurQ-HI with the substrate analogue showed that Glu89 was located in the proximity of both the C2 atom and the oxygen at the C3 position of the bound inhibitor and that no other potential acid/base residue that could act as an active site acid/base was located in the vicinity. The conserved residues Glu120 and Lys239 were found within hydrogen bonding distance of the C5 hydroxyl group and C6 phosphate group, suggesting that they play a role in substrate binding and ring opening. Combining these results with previous biochemical data, we propose a one-base mechanism of action in which Glu89 functions to both deprotonate at the C2 position and assist in the departure of the lactyl ether at the C3 position. This same residue would serve to deprotonate the incoming water and reprotonate the enolate in the second half of the catalytic cycle.

Deamination of 6-aminodeoxyfutalosine in menaquinone biosynthesis by distantly related enzymes.

Proteins of unknown function belonging to cog1816 and cog0402 were characterized. Sav2595 from Steptomyces avermitilis MA-4680, Acel0264 from Acidothermus cellulolyticus 11B, Nis0429 from Nitratiruptor sp. SB155-2 and Dr0824 from Deinococcus radiodurans R1 were cloned, purified, and their substrate profiles determined. These enzymes were previously incorrectly annotated as adenosine deaminases or chlorohydrolases. It was shown here that these enzymes actually deaminate 6-aminodeoxyfutalosine. The deamination of 6-aminodeoxyfutalosine is part of an alternative menaquinone biosynthetic pathway that involves the formation of futalosine. 6-Aminodeoxyfutalosine is deaminated by these enzymes with catalytic efficiencies greater than 10(5) M(-1) s(-1), Km values of 0.9-6.0 µM, and kcat values of 1.2-8.6 s(-1). Adenosine, 2'-deoxyadenosine, thiomethyladenosine, and S-adenosylhomocysteine are deaminated at least an order of magnitude slower than 6-aminodeoxyfutalosine. The crystal structure of Nis0429 was determined and the substrate, 6-aminodeoxyfutalosine, was positioned in the active site on the basis of the presence of adventitiously bound benzoic acid. In this model, Ser-145 interacts with the carboxylate moiety of the substrate. The structure of Dr0824 was also determined, but a collapsed active site pocket prevented docking of substrates. A computational model of Sav2595 was built on the basis of the crystal structure of adenosine deaminase and substrates were docked. The model predicted a conserved arginine after ß-strand 1 to be partially responsible for the substrate specificity of Sav2595.

Structure/function analysis of Pasteurella multocida heparosan synthases: toward defining enzyme specificity and engineering novel catalysts.

The Pasteurella multocida heparosan synthases, PmHS1 and PmHS2, are homologous (∼65% identical) bifunctional glycosyltransferase proteins found in Type D Pasteurella. These unique enzymes are able to generate the glycosaminoglycan heparosan by polymerizing sugars to form repeating disaccharide units from the donor molecules UDP-glucuronic acid (UDP-GlcUA) and UDP-N-acetylglucosamine (UDP-GlcNAc). Although these isozymes both generate heparosan, the catalytic phenotypes of these isozymes are quite different. Specifically, during in vitro synthesis, PmHS2 is better able to generate polysaccharide in the absence of exogenous acceptor (de novo synthesis) than PmHS1. Additionally, each of these enzymes is able to generate polysaccharide using unnatural sugar analogs in vitro, but they exhibit differences in the substitution patterns of the analogs they will employ. A series of chimeric enzymes has been generated consisting of various portions of both of the Pasteurella heparosan synthases in a single polypeptide chain. In vitro radiochemical sugar incorporation assays using these purified chimeric enzymes have shown that most of the constructs are enzymatically active, and some possess novel characteristics including the ability to produce nearly monodisperse polysaccharides with an expanded range of sugar analogs. Comparison of the kinetic properties and the sequences of the wild-type enzymes with the chimeric enzymes has enabled us to identify regions that may be responsible for some aspects of both donor binding specificity and acceptor usage. In combination with previous work, these approaches have enabled us to better understand the structure/function relationship of this unique family of glycosyltransferases.

Potential rearrangements in the reaction catalyzed by the indole prenyltransferase FtmPT1.

The indole prenyltransferase FtmPT1 catalyzes the C-2 normal prenylation of brevianamide F (cyclo-L-Trp-L-Pro) to give tryprostatin B. A previous structural analysis and studies with alternate substrates suggest that the reaction might not proceed through a direct C-2 attack, but could involve a C-3 prenylation followed by a rearrangement. In this work we investigated the reactivity of FtmPT1 with tryptophan, 5-hydroxybrevianamide, and 2-methylbrevianamide, and isolated products that had been reverse prenylated at C-3 and normal prenylated at N-1, C-3, or C-4. The formation of these products can be rationalized through mechanisms involving either an initial C-3 normal or C-3 reverse prenylation. In addition, we demonstrate that a C-3 reverse prenylated indole can undergo a nonenzymatic aza-Cope rearrangement at 37 °C to give an N-1 normal prenylated product. Together, these studies broaden the known product scope of this interesting catalyst and suggest that alternative mechanisms might be operating.