Rethinking Law and Gospel in the Way We Do Preaching

This paper evaluates the impact of C. F. W. Walther’s Law and Gospel and Richard Caemmerer’s goal, malady, means approach to homiletics, also discussing the potential trap of law-gospel reductionism. A suggested pathway forward is a reemphasis on a creedal approach to Lutheran theology and preaching as well as a renewal of rhetoric as foundational to ultimately restoring a positive view of the third use or function of the law in Lutheran preaching. Having done so, the reader may certainly apply this positive view of the law as it relates to preaching on other topics related to the Christian Life including justice, compassion, and race relations

Biosynthesis of the sactipeptide Ruminococcin C by the human microbiome: Mechanistic insights into thioether bond formation by radical SAM enzymes

Despite its major importance in human health, the metabolic potential of the human gut microbiota is still poorly understood. We have recently shown that biosynthesis of Ruminococcin C (RumC), a novel ribosomally synthesized and posttranslationally modified peptide (RiPP) produced by the commensal bacterium Ruminococcus gnavus, requires two radical SAM enzymes (RumMC1 and RumMC2) catalyzing the formation of four C-alpha-thioether bridges. These bridges, which are essential for RumC's antibiotic properties against human pathogens such as Clostridium perfringens, define two hairpin domains giving this sactipeptide (sulfur-to-alpha-carbon thioether-containing peptide) an unusual architecture among natural products. We report here the biochemical and spectroscopic characterizations of RumMC2. EPR spectroscopy and mutagenesis data support that RumMC2 is a member of the large family of SPASM domain radical SAM enzymes characterized by the presence of three [4Fe-4S] clusters. We also demonstrate that this enzyme initiates its reaction by C-alpha H-atom abstraction and is able to catalyze the formation of nonnatural thioether bonds in engineered peptide substrates. Unexpectedly, our data support the formation of a ketoimine rather than an alpha,beta-dehydro-amino acid intermediate during C-alpha-thioether bridge LC-MS/MS fragmentation. Finally, we explored the roles of the leader peptide and of the RiPP precursor peptide recognition element, present in myriad RiPP-modifying enzymes. Collectively, our data support a more complex role for the peptide recognition element and the core peptide for the installation of posttranslational modifications in RiPPs than previously anticipated and suggest a possible reaction intermediate for thioether bond formation

Extracorporeal Membrane Oxygenation for Severe Acute Respiratory Distress Syndrome associated with COVID-19: An Emulated Target Trial Analysis.

RATIONALE: Whether COVID patients may benefit from extracorporeal membrane oxygenation (ECMO) compared with conventional invasive mechanical ventilation (IMV) remains unknown. OBJECTIVES: To estimate the effect of ECMO on 90-Day mortality vs IMV only Methods: Among 4,244 critically ill adult patients with COVID-19 included in a multicenter cohort study, we emulated a target trial comparing the treatment strategies of initiating ECMO vs. no ECMO within 7 days of IMV in patients with severe acute respiratory distress syndrome (PaO2/FiO2 <80 or PaCO2 ≥60 mmHg). We controlled for confounding using a multivariable Cox model based on predefined variables. MAIN RESULTS: 1,235 patients met the full eligibility criteria for the emulated trial, among whom 164 patients initiated ECMO. The ECMO strategy had a higher survival probability at Day-7 from the onset of eligibility criteria (87% vs 83%, risk difference: 4%, 95% CI 0;9%) which decreased during follow-up (survival at Day-90: 63% vs 65%, risk difference: -2%, 95% CI -10;5%). However, ECMO was associated with higher survival when performed in high-volume ECMO centers or in regions where a specific ECMO network organization was set up to handle high demand, and when initiated within the first 4 days of MV and in profoundly hypoxemic patients. CONCLUSIONS: In an emulated trial based on a nationwide COVID-19 cohort, we found differential survival over time of an ECMO compared with a no-ECMO strategy. However, ECMO was consistently associated with better outcomes when performed in high-volume centers and in regions with ECMO capacities specifically organized to handle high demand. This article is open access and distributed under the terms of the Creative Commons Attribution Non-Commercial No Derivatives License 4.0 (http://creativecommons.org/licenses/by-nc-nd/4.0/)

Thioether bond formation by SPASM domain radical SAM enzymes: C α H-atom abstraction in subtilosin A biosynthesis

International audienceAlbA is a radical SAM enzyme catalyzing the formation of three unusual thioether bonds in the antibiotic subtilosin A. We demonstrate here that AlbA catalyzes direct Cα H-atom abstraction and likely contains three essential [4Fe-4S] centers. This leads us to propose novel mechanistic perspectives for thioether bond catalysis by radical SAM enzymes

Biosynthetic Versatility and Coordinated Action of 5′-Deoxyadenosyl Radicals in Deazaflavin Biosynthesis

Coenzyme F₄₂₀ is a redox cofactor found in methanogens and in various actinobacteria. Despite the major biological importance of this cofactor, the biosynthesis of its deazaflavin core (8-hydroxy-5-deazaflavin, F_o) is still poorly understood. F_o synthase, the enzyme involved, is an unusual multidomain radical SAM enzyme that uses two separate 5′-deoxyadenosyl radicals to catalyze F_o formation. In this paper, we report a detailed mechanistic study on this complex enzyme that led us to identify (1) the hydrogen atoms abstracted from the substrate by the two radical SAM domains, (2) the second tyrosine-derived product, (3) the reaction product of the CofH-catalyzed reaction, (4) the demonstration that this product is a substrate for CofG, and (5) a stereochemical study that is consistent with the formation of a <i>p</i>-hydroxybenzyl radical at the CofH active site. These results enable us to propose a mechanism for F_o synthase and uncover a new catalytic motif in radical SAM enzymology involving the use of two 5′-deoxyadenosyl radicals to mediate the formation of a complex heterocycle

Mechanistic Investigations of PoyD, a Radical S -Adenosyl- l -methionine Enzyme Catalyzing Iterative and Directional Epimerizations in Polytheonamide A Biosynthesis

International audienceRibosomally synthesized and post-translationally modified peptides (RiPPs) are a growing family of bioactive peptides. Among RiPPs, the bacterial toxin polytheonamide A is characterized by a unique set of post-translational modifications catalyzed by novel radical S-adenosyl-l-methionine (SAM) enzymes. Here we show that the radical SAM enzyme PoyD catalyzes in vitro polytheonamide epimerization in a C-to-N directional manner. By combining mutagenesis experiments with labeling studies and investigating the enzyme substrate promiscuity, we deciphered in detail the mechanism of PoyD. We notably identified a critical cysteine residue as a likely key H atom donor and demonstrated that PoyD belongs to a distinct family of radical SAM peptidyl epimerases. In addition, our study shows that the core peptide directly influences the epimerization pattern allowing for production of peptides with unnatural epimerization patterns

One enzyme for two radical reactions: Deciphering the mechanism of a novel radical SAM enzyme critical for F-420 cofactor biosynthesis

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Biosynthesis of F₀, Precursor of the F₄₂₀ Cofactor, Requires a Unique Two Radical-SAM Domain Enzyme and Tyrosine as Substrate

Cofactors play key roles in metabolic pathways. Among them F₄₂₀ has proved to be a very attractive target for the selective inhibition of archaea and actinobacteria. Its biosynthesis, in a unique manner, involves a key enzyme, F₀-synthase. This enzyme is a large monomer in actinobacteria, while it is constituted of two subunits in archaea and cyanobacteria. We report here the purification of both types of F₀-synthase and their <i>in vitro</i> activities. Our study allows us to establish that F₀-synthase, from both types, uses 5-amino-6-ribitylamino-2,4­(1<i>H</i>,3<i>H</i>)-pyrimidinedione and tyrosine as substrates but not 4-hydroxylphenylpyruvate as previously suggested. Furthermore, our data support the fact that F₀-synthase generates two 5′-deoxyadenosyl radicals for catalysis which is unprecedented in reaction catalyzed by radical SAM enzymes