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.

Investigating the Relationship between Fear of Failure and the Delivery of End-of-Life Care: A Questionnaire Study.

OBJECTIVE: To investigate whether fear of failure (FOF) influences a clinician's perception of how confident and comfortable they are in their delivery of end-of-life (EOL) care. METHODS: Cross-sectional questionnaire study with recruitment of physicians and nurses across two large NHS hospital trusts in the UK and national UK professional networks. A total of 104 physicians and 101 specialist nurses across 20 hospital specialities provided data that were analysed using a two-step hierarchical regression. RESULTS: The study validated the PFAI measure for use in medical contexts. Number of EOL conversations, gender, and role were shown to impact confidence and comfortableness with EOL care. Four FOF subscales did show a significant relationship with perceived delivery of EOL care. CONCLUSION: Aspects of FOF can be shown to negatively impact the clinician experience of delivering EOL care. CLINICAL IMPLICATIONS: Further study should explore how FOF develops, populations that are more susceptible, sustaining factors, and its impact on clinical care. Techniques developed to manage FOF in other populations can now be investigated in a medical population.

Identification of a poly-cyclopropylglycine-containing peptide via bioinformatic mapping of radical S-adenosylmethionine enzymes.

Peptide-derived natural products are a large class of bioactive molecules that often contain chemically challenging modifications. In the biosynthesis of ribosomally synthesized and posttranslationally modified peptides (RiPPs), radical-SAM (rSAM) enzymes have been shown to catalyze the formation of ether, thioether, and carbon-carbon bonds on the precursor peptide. The installation of these bonds typically establishes the skeleton of the mature RiPP. To facilitate the search for unexplored rSAM-dependent RiPPs for the community, we employed a bioinformatic strategy to screen a subfamily of peptide-modifying rSAM enzymes which are known to bind up to three [4Fe-4S] clusters. A sequence similarity network was used to partition related families of rSAM enzymes into >250 clusters. Using representative sequences, genome neighborhood diagrams were generated using the Genome Neighborhood Tool. Manual inspection of bacterial genomes yielded numerous putative rSAM-dependent RiPP pathways with unique features. From this analysis, we identified and experimentally characterized the rSAM enzyme, TvgB, from the tvg gene cluster from Halomonas anticariensis. In the tvg gene cluster, the precursor peptide, TvgA, is comprised of a repeating TVGG motif. Structural characterization of the TvgB product revealed the repeated formation of cyclopropylglycine, where a new bond is formed between the γ-carbons on the precursor valine. This novel RiPP modification broadens the functional potential of rSAM enzymes and validates the proposed bioinformatic approach as a practical broad search tool for the discovery of new RiPP topologies.

Physician emotional experience of communication and decision making with end-of-life patients: qualitative studies systematic review.

OBJECTIVE: To explore the emotional experience of physicians in acute settings when encountering end-of-life conversations and decision making. METHOD: Thematic synthesis of qualitative studies. Medline, PsychInfo, PubMed, BNI and CIAHL were searched from 1985 to 2021 for studies published in English. Data extraction was informed by a framework created for assessing methodological quality by Polanin, Pigott, Espelage and Grotpeter (2019) and adapted by Draper et al. (2019). RESULTS: Of 8429 papers identified, 17 were selected for review. Two themes containing 10 subthemes described the emotional and psychological factors impacting the experience of end-of-life care, namely: a tension between desire and ability to communicate end-of-life news, and a conflict of hiding versus revealing self across several practical and emotional contexts. CONCLUSION: Medical training is only a small factor in how well a person copes with end-of-life care and may sometimes feed negative appraisals . Lack of support from senior colleagues, fear of criticism and a sense of perceived failure were linked to lower self-efficacy in end-of-life care. Beyond learning practical skills, physicians benefit from understanding the psychological factors impacting their experience and in building self-efficacy, and observing senior colleagues effectively process strong and difficult emotions. PRACTICAL IMPLICATIONS: Promoting personal reflection and sharing of the experiences encountered in end-of-life care, especially modelled from senior colleagues, may contribute to improvements in competence and reduce the impact of heroism, feelings of failure and avoidance in practice.

How a Subfamily of Radical S-Adenosylmethionine Enzymes Became a Mainstay of Ribosomally Synthesized and Post-translationally Modified Peptide Discovery.

Radical S-adenosylmethionine (rSAM) enzymes are a large and diverse superfamily of enzymes, some of which are known to participate in the biosynthesis of ribosomally synthesized and post-translationally modified peptides (RiPPs). Specifically, a subfamily of rSAM proteins with an elongated C-terminus known as a SPASM domain have become a fixation in the discovery of new RiPP natural products. Arguably, a structural study, a bioinformatic study, and a functional study built the foundation of the research for rSAM-SPASM-protein-modified RiPPs. In this Review, we focus on these three studies and how they initiated what has become an increasingly productive field. In addition, we discuss the current state of RiPPs that depends on rSAM-SPASM proteins and provide guidelines to consider in future research. Lastly, we discuss how genome mining tools have become a powerful means to identify and predict new RiPP natural products. Despite the state of our current knowledge, we do not completely understand the relationship of rSAM-SPASM chemistry, substrate recognition, and the structure-function relationship as it pertains to RiPP biosynthesis, and as such, there remain many interesting findings waiting to be discovered in the future.

Biosynthesis of the redox cofactor mycofactocin is controlled by the transcriptional regulator MftR and induced by long-chain acyl-CoA species.

Mycofactocin (MFT) is a ribosomally synthesized and post-translationally-modified redox cofactor found in pathogenic mycobacteria. While MFT biosynthetic proteins have been extensively characterized, the physiological conditions under which MFT biosynthesis is required are not well understood. To gain insights into the mechanisms of regulation of MFT expression in Mycobacterium smegmatis mc2155, we investigated the DNA-binding and ligand-binding activities of the putative TetR-like transcription regulator, MftR. In this study, we demonstrated that MftR binds to the mft promoter region. We used DNase I footprinting to identify the 27 bp palindromic operator located 5' to mftA and found it to be highly conserved in Mycobacterium tuberculosis, Mycobacterium bovis, Mycobacterium ulcerans, and Mycobacterium marinum. To determine under which conditions the mft biosynthetic gene cluster (BGC) is induced, we screened for effectors of MftR. As a result, we found that MftR binds to long-chain acyl-CoAs with low micromolar affinities. To demonstrate that oleoyl-CoA induces the mft BGC in vivo, we re-engineered a fluorescent protein reporter system to express an MftA-mCherry fusion protein. Using this mCherry fluorescent readout, we show that the mft BGC is upregulated in M. smegmatis mc2155 when oleic acid is supplemented to the media. These results suggest that MftR controls expression of the mft BGC and that MFT production is induced by long-chain acyl-CoAs. Since MFT-dependent dehydrogenases are known to colocalize with acyl carrier protein/CoA-modifying enzymes, these results suggest that MFT might be critical for fatty acid metabolism or cell wall reorganization.

Clinical Immunogenicity Evaluation of Eptinezumab, a Therapeutic Humanized Monoclonal Antibody Targeting Calcitonin Gene-Related Peptide (CGRP) for the Preventive Treatment of Migraine.

Background: Eptinezumab is a humanized monoclonal antibody that selectively binds calcitonin gene-related peptide and is indicated for the preventive treatment of migraine in adults. This analysis characterizes the immunogenic profile of eptinezumab using data from clinical trials of eptinezumab for migraine prevention. Methods: Immunogenicity data were collected from five studies that included 2076 patients with episodic or chronic migraine treated with eptinezumab at dose levels ranging from 10 to 1000 mg, administered intravenously for up to 4 doses at 12-week intervals. Anti-drug antibody (ADA) results were available from 2074 of these patients. Four studies were randomized, double-blind, placebo-controlled trials with ADA monitoring for up to 56 weeks; one was a 2-year, open-label, phase 3 safety study with ADA monitoring for 104 weeks. Patients who had a confirmed ADA-positive result at the end-of-study visit were monitored for up to 6 additional months. Development of ADA and neutralizing antibodies (NAbs) were evaluated to explore three key areas of potential impact: pharmacokinetic exposure profile (eptinezumab trough plasma concentrations), efficacy (change in monthly migraine days), and safety (rates of treatment-emergent adverse events). These studies included methods designed to capture the dynamics of a potential humoral immune response to eptinezumab treatment, and descriptive analyses were applied to interpret the relationship of ADA signals to drug exposure, efficacy, and safety. Results: Pooled across the five clinical trials, treatment-emergent ADAs and NAbs occurred in 15.8 and 6.2% of eptinezumab-treated patients, respectively. Highly consistent profiles were observed across all studies, with initial onset of detectable ADA observed at the week 8 measurement and maximal ADA frequency and titer observed at week 24, regardless of eptinezumab dose level or number of doses. After 24 weeks, the ADA and NAb titers steadily declined despite additional doses of eptinezumab. Interpretation: Collectively, these integrated analyses did not demonstrate any clinically meaningful impact from ADA occurring after treatment with eptinezumab. The ADA profiles were low titer and transient, with the incidence and magnitude of ADA or NAb responses declining after week 24. Development of ADAs and NAbs did not impact the efficacy and safety profiles of eptinezumab.

New developments in RiPP discovery, enzymology and engineering.

Covering: up to June 2020Ribosomally-synthesized and post-translationally modified peptides (RiPPs) are a large group of natural products. A community-driven review in 2013 described the emerging commonalities in the biosynthesis of RiPPs and the opportunities they offered for bioengineering and genome mining. Since then, the field has seen tremendous advances in understanding of the mechanisms by which nature assembles these compounds, in engineering their biosynthetic machinery for a wide range of applications, and in the discovery of entirely new RiPP families using bioinformatic tools developed specifically for this compound class. The First International Conference on RiPPs was held in 2019, and the meeting participants assembled the current review describing new developments since 2013. The review discusses the new classes of RiPPs that have been discovered, the advances in our understanding of the installation of both primary and secondary post-translational modifications, and the mechanisms by which the enzymes recognize the leader peptides in their substrates. In addition, genome mining tools used for RiPP discovery are discussed as well as various strategies for RiPP engineering. An outlook section presents directions for future research.

Pharmacologic Characterization of ALD403, a Potent Neutralizing Humanized Monoclonal Antibody Against the Calcitonin Gene-Related Peptide.

ALD403 is a genetically engineered, humanized immunoglobulin G1 monoclonal antibody that inhibits the action of human calcitonin gene-related peptide (CGRP). Clinical trial data indicate that ALD403 is effective as a preventive therapy for migraine and has an acceptable safety profile. For preclinical characterization of ALD403, rabbit antibodies targeting α-CGRP were humanized and modified to eliminate fragment crystallizable (Fc) γ receptor (FcγR) and complement interactions. The ability of ALD403 to inhibit CGRP-induced cAMP production was assessed using a cAMP bioassay (Meso Scale Discovery). The IC50 for inhibition of cAMP release was 434 and 288 pM with the rabbit-human chimera antibody and the humanized ALD403, respectively. ALD403 inhibited α-CGRP binding with an IC50 of 4.7 × 10-11 and 1.2 × 10-10 M for the α-CGRP and AMY1 receptors, respectively. ALD403 did not induce antibody-dependent cellular cytotoxicity or complement-dependent cytotoxicity and did not stably interact with any of the FcγR mediating these functions, exhibiting only weak binding to FcγRI. ALD403 significantly lowered capsaicin-induced blood flow responses in rodents at all time points starting at 5 minutes postapplication in a dose-dependent manner. In conclusion, ALD403 is a potent functional ligand inhibitor of α-CGRP‒driven pharmacology. SIGNIFICANCE STATEMENT: α-Calcitonin gene-related peptide blockade by ALD403 was assessed via radiolabeled ligand displacement, in vitro inhibition of cell signaling, and in vivo inhibition of capsaicin-induced vasodilation. Lack of engagement of fragment crystallizable-mediated immune-effector functions by ALD403 was shown.

Population pharmacokinetic and exposure-response analysis of eptinezumab in the treatment of episodic and chronic migraine.

Eptinezumab is a humanized mAb that targets calcitonin gene-related peptide and is under regulatory review for the prevention of episodic and chronic migraine (EM, CM). It is important to determine whether exposures achieved with intravenous (IV) administration of eptinezumab achieve desired pharmacologic effects. Population pharmacokinetics, including dose- and exposure-response analyses, were performed using patient-level data from the eptinezumab clinical trial program with IV doses ranging from 10 to 1000 mg in pharmacokinetic analyses or 10 to 300 mg in phase 2/3 clinical studies in patients with EM or CM. Exposure-response analysis explored the relationship between eptinezumab exposure metrics and efficacy parameters including monthly migraine days. The pharmacokinetic profile of eptinezumab was characterized by rapid attainment of maximum plasma concentration (ie, end of IV administration) and a terminal half-life of 27 days. Covariate analysis found that patient characteristics had no clinically significant effects on pharmacokinetic parameters and were insufficient to influence dosing. Dose- and exposure-response analyses found exposure with single doses ≥100 mg was associated with greater efficacy compared with doses ≤30 mg and a plateau of effect between 100 and 300 mg. A saturable inhibitory Emax model found the exposure over 12 weeks produced by single-dose eptinezumab 100 and 300 mg exceeded the exposure estimates required to achieve 90% of the maximal efficacy (EC90 ). This pharmacokinetic analysis of eptinezumab supports dosing every 12 weeks with no adjustment for patient characteristics, including exposures associated with 100- or 300-mg doses producing optimal efficacy effects. The similar efficacy profiles support 100 mg as the lowest effective dose of eptinezumab.

Structural insights for vanadium catecholates and iron­sulfur clusters obtained from multiple data analysis methods applied to electron spin relaxation data.

Electron paramagnetic resonance (EPR) inversion recovery curves for vanadium catecholates and iron­sulfur clusters were analyzed with three models: the sum of two exponentials, a stretched exponential, and a model-free distribution of exponentials (UPEN). For all data sets studied fits with a stretched exponential were statistically indistinguishable from the sum of two exponentials, and were significantly better than for single exponentials. UPEN provides insights into the structures of the distributions. For a vanadium(IV) tris catecholate the distribution of relaxation rates calculated with UPEN shows the contribution from spectral diffusion at low temperatures. The energy of the local mode for this complex, found from the temperature dependence of the spin lattice relaxation, is consistent with values expected for a metal-ligand vibration. For the [2Fe-2S]+ cluster in pyruvate formate lyase activating enzyme (PFL-AE) the small stretched exponential ß values (0.3) at low temperature and the distributions calculated with UPEN reflect the contribution from a second rapidly relaxing species that could be difficult to detect by continuous wave EPR. The distributions in 1/T1 for the [4Fe-4S]+ clusters in Mycofactocin maturase were about a factor of four wider than for the three other systems studied. The very broad distribution of relaxation rates may be due to protein mobility and distributions in electronic energies and local environments for the clusters. UPEN provides insight into several situations that can result in low values of stretch parameter ß including contributions from spectral diffusion, overlapping signals from distinguishable clusters, or very wide distributions.

MftD Catalyzes the Formation of a Biologically Active Redox Center in the Biosynthesis of the Ribosomally Synthesized and Post-translationally Modified Redox Cofactor Mycofactocin.

Mycofactocin (MFT) is a putative ribosomally synthesized and post-translationally modified (RiPP) redox cofactor. The biosynthesis of MFT is encoded by the gene cluster mftABCDEF. While processing of the precursor peptide by MftB, MftC, and MftE has been shown to result in the formation of the small molecule 3-amino-5-[(p-hydroxyphenyl)methyl]-4,4-dimethyl-2-pyrrolidinone (AHDP), no activity has been shown for the putative dehydrogenase MftD and the putative glycosyltransferase MftF. In addition, evidence demonstrating that MFT is a redox cofactor has only been limited to the requirement of mft genes for ethanol assimilation in Mycobacterium smegmatis mc2155. Here, we demonstrate that MftD catalyzes the oxidative deamination of AHDP, forming an α-keto moiety on the resulting molecule, which we call pre-mycofactocin (PMFT). We characterize PMFT by 1D and 2D NMR spectroscopy techniques and by high-resolution mass spectrometry data to solve its structure. We further characterized PMFT by cyclic voltammetry and found its midpoint potential to be ∼255 mV. Lastly, we demonstrate that PMFT is a biologically active redox cofactor that oxidizes NADH bound by M. smegmatis carveol dehydrogenase (MsCDH) and can be used by MsCDH in the oxidation of carveol. These data demonstrate for the first time that PMFT functions as a biologically active redox mediator and provides the most direct evidence to date that MFT is a RiPP-derived redox cofactor.

A two-component protease in Methylorubrum extorquens with high activity toward the peptide precursor of the redox cofactor pyrroloquinoline quinone.

Pyrroloquinoline quinone is a prominent redox cofactor in many prokaryotes, produced from a ribosomally synthesized and post-translationally modified peptide PqqA via a pathway comprising four conserved proteins PqqB-E. These four proteins are now fairly well-characterized and span radical SAM activity (PqqE), aided by a peptide chaperone (PqqD), a dual hydroxylase (PqqB), and an eight-electron, eight-proton oxidase (PqqC). A full description of this pathway has been hampered by a lack of information regarding a protease/peptidase required for the excision of an early, cross-linked di-amino acid precursor to pyrroloquinoline quinone. Herein, we isolated and characterized a two-component heterodimer protein from the α-proteobacterium Methylobacterium (Methylorubrum) extorquens that can rapidly catalyze cleavage of PqqA into smaller peptides. Using pulldown assays, surface plasmon resonance, and isothermal calorimetry, we demonstrated the formation of a complex PqqF/PqqG, with a KD of 300 nm We created a molecular model of the heterodimer by comparison with the Sphingomonas sp. A1 M16B Sph2681/Sph2682 protease. Analysis of time-dependent patterns for the appearance of proteolysis products indicates high specificity of PqqF/PqqG for serine side chains. We hypothesize that PqqF/PqqG initially cleaves between the PqqE/PqqD-generated cross-linked form of PqqA, with nonspecific cellular proteases completing the release of a suitable substrate for the downstream enzyme PqqB. The finding of a protease that specifically targets serine side chains is rare, and we propose that this activity may be useful in proteomic analyses of the large family of proteins that have undergone post-translational phosphorylation at serine.

Discovery of Hydroxylase Activity for PqqB Provides a Missing Link in the Pyrroloquinoline Quinone Biosynthetic Pathway.

Understanding the biosynthesis of cofactors is fundamental to the life sciences, yet to date a few important pathways remain unresolved. One example is the redox cofactor pyrroloquinoline quinone (PQQ), which is critical for C1 metabolism in many microorganisms, a disproportionate number of which are opportunistic human pathogens. While the initial and final steps of PQQ biosynthesis, involving PqqD/E and PqqC, have been elucidated, the precise nature and order of the remaining transformations in the pathway are unknown. Here we show evidence that the remaining essential biosynthetic enzyme PqqB is an iron-dependent hydroxylase catalyzing oxygen-insertion reactions that are proposed to produce the quinone moiety of the mature PQQ cofactor. The demonstrated reactions of PqqB are unprecedented within the metallo ß-lactamase protein family and expand the catalytic repertoire of nonheme iron hydroxylases. These new findings also generate a nearly complete description of the PQQ biosynthetic pathway.

Occurrence, function, and biosynthesis of mycofactocin.

Mycofactocin is a member of the rapidly growing class of ribosomally synthesized and post-translationally modified peptide (RiPP) natural products. Although the mycofactocin biosynthetic pathway is widely distributed among Mycobacterial species, the structure, function, and biosynthesis of the pathway product remain unknown. This mini-review will discuss the current state of knowledge regarding the mycofactocin biosynthetic pathway. In particular, we focus on the architecture and distribution of the mycofactocin biosynthetic cluster, mftABCDEF, among the Actinobacteria phylum. We discuss the potential molecular and physiological role of mycofactocin. We review known biosynthetic steps involving MftA, MftB, MftC, and MftE and relate them to pyrroloquinoline quinone biosynthesis. Lastly, we propose the function of the remaining putative biosynthetic enzymes, MftD and MftF.

Spectroscopic and Electrochemical Characterization of the Mycofactocin Biosynthetic Protein, MftC, Provides Insight into Its Redox Flipping Mechanism.

Mycofactocin is a putative redox cofactor and is classified as a ribosomally synthesized and post-translationally modified peptide (RiPP). Some RiPP natural products, including mycofactocin, rely on a radical S-adenosylmethionine (RS, SAM) protein to modify the precursor peptide. Mycofactocin maturase, MftC, is a unique RS protein that catalyzes the oxidative decarboxylation and C-C bond formation on the precursor peptide MftA. However, the number, chemical nature, and catalytic roles for the MftC [Fe-S] clusters remain unknown. Here, we report that MftC binds a RS [4Fe-4S] cluster and two auxiliary [4Fe-4S] clusters that are required for MftA modification. Furthermore, electron paramagnetic resonance spectra of MftC suggest that SAM and MftA affect the environments of the RS and Aux I cluster, whereas the Aux II cluster is unaffected by the substrates. Lastly, reduction potential assignments of individual [4Fe-4S] clusters by protein film voltammetry show that their potentials are within 100 mV of each other.

Pharmacologic Characterization of ALD1910, a Potent Humanized Monoclonal Antibody against the Pituitary Adenylate Cyclase-Activating Peptide.

Migraine is a debilitating disease that affects almost 15% of the population worldwide and is the first cause of disability in people under 50 years of age, yet its etiology and pathophysiology remain incompletely understood. Recently, small molecules and therapeutic antibodies that block the calcitonin gene-related peptide (CGRP) signaling pathway have reduced migraine occurrence and aborted acute attacks of migraine in clinical trials and provided prevention in patients with episodic and chronic migraine. Heterogeneity is present within each diagnosis and patient's response to treatment, suggesting migraine as a final common pathway potentially activated by multiple mechanisms, e.g., not all migraine attacks respond to or are prevented by anti-CGRP pharmacological interventions. Consequently, other unique mechanisms central to migraine pathogenesis may present new targets for drug development. Pituitary adenylate cyclase-activating peptide (PACAP) is an attractive novel target for treatment of migraines. We generated a specific, high-affinity, neutralizing monoclonal antibody (ALD1910) with reactivity to both PACAP38 and PACAP27. In vitro, ALD1910 effectively antagonizes PACAP38 signaling through the pituitary adenylate cyclase-activating peptide type I receptor, vasoactive intestinal peptide receptor 1, and vasoactive intestinal peptide receptor 2. ALD1910 recognizes a nonlinear epitope within PACAP and blocks its binding to the cell surface. To test ALD1910 antagonistic properties directed against endogenous PACAP, we developed an umbellulone-induced rat model of neurogenic vasodilation and parasympathetic lacrimation. In vivo, this model demonstrates that the antagonistic activity of ALD1910 is dose-dependent, retaining efficacy at doses as low as 0.3 mg/kg. These results indicate that ALD1910 represents a potential therapeutic antibody to address PACAP-mediated migraine.

Mycofactocin Biosynthesis Proceeds through 3-Amino-5-[( p-hydroxyphenyl)methyl]-4,4-dimethyl-2-pyrrolidinone (AHDP); Direct Observation of MftE Specificity toward MftA.

The structure of the ribosomally synthesized and post-translationally modified peptide product mycofactocin is unknown. Recently, the first step in mycofactocin biosynthesis was shown to be catalyzed by MftC in two S-adenosylmethionine-dependent steps. In the first step, MftC catalyzes the oxidative decarboxylation of the MftA peptide to produce the styrene-containing intermediate MftA**, followed by a subsequent C-C bond formation to yield the lactam-containing MftA*. Here, we demonstrate the subsequent biosynthetic step catalyzed by MftE is specific for MftA*. The hydrolysis of MftA* leads to the formation of MftA(1-28) and 3-amino-5-[( p-hydroxyphenyl)methyl]-4,4-dimethyl-2-pyrrolidinone (AHDP). The hydrolysis reaction is Fe2+-dependent, and addition of the metal to the reaction mixture leads to a kobs of ∼0.2 min-1. Lastly, we validate the structure of AHDP by 1H, 13C, and COSY nuclear magnetic resonance techniques as well as mass spectrometry.

X-ray and EPR Characterization of the Auxiliary Fe-S Clusters in the Radical SAM Enzyme PqqE.

The Radical SAM (RS) enzyme PqqE catalyzes the first step in the biosynthesis of the bacterial cofactor pyrroloquinoline quinone, forming a new carbon-carbon bond between two side chains within the ribosomally synthesized peptide substrate PqqA. In addition to the active site RS 4Fe-4S cluster, PqqE is predicted to have two auxiliary Fe-S clusters, like the other members of the SPASM domain family. Here we identify these sites and examine their structure using a combination of X-ray crystallography and Mössbauer and electron paramagnetic resonance (EPR) spectroscopies. X-ray crystallography allows us to identify the ligands to each of the two auxiliary clusters at the C-terminal region of the protein. The auxiliary cluster nearest the RS site (AuxI) is in the form of a 2Fe-2S cluster ligated by four cysteines, an Fe-S center not seen previously in other SPASM domain proteins; this assignment is further supported by Mössbauer and EPR spectroscopies. The second, more remote cluster (AuxII) is a 4Fe-4S center that is ligated by three cysteine residues and one aspartate residue. In addition, we examined the roles these ligands play in catalysis by the RS and AuxII clusters using site-directed mutagenesis coupled with EPR spectroscopy. Lastly, we discuss the possible functional consequences that these unique AuxI and AuxII clusters may have in catalysis for PqqE and how these may extend to additional RS enzymes catalyzing the post-translational modification of ribosomally encoded peptides.