scholarly journals Thioether bond formation by SPASM domain radical SAM enzymes: Cα H-atom abstraction in subtilosin A biosynthesis

2016 ◽  
Vol 52 (37) ◽  
pp. 6249-6252 ◽  
Author(s):  
Alhosna Benjdia ◽  
Alain Guillot ◽  
Benjamin Lefranc ◽  
Hubert Vaudry ◽  
Jérôme Leprince ◽  
...  
Keyword(s):  
Bond Formation ◽  
Radical Sam ◽  
Radical Sam Enzymes ◽  
Radical Sam Enzyme ◽  
Synthetic Approaches

The radical SAM enzyme AlbA has been reported to catalyze the formation of a thioether bond in the antibiotic subtilosin A. By modeling, biochemical and synthetic approaches, we propose novel mechanistic perspectives on this emerging group of enzymes.

scholarly journals Accumulation of an unprecedented 5′-deoxyadenos-4′-yl radical unmasks the kinetics of the radical-mediated C-C bond formation step in MoaA catalysis

2020 ◽  
Author(s):  
Haoran Pang ◽  
Edward A. Lilla ◽  
Pan Zhang ◽  
Du Zhang ◽  
Thomas P. Shields ◽  
...  
Keyword(s):  
Biochemical Characterization ◽  
Bond Formation ◽  
Kinetic Characterization ◽  
Amino Acid Residues ◽  
Radical Sam ◽  
Radical Sam Enzymes ◽  
Rate Acceleration ◽  
Radical Sam Enzyme ◽  
Radical Catalysis

AbstractRadical S-adenosyl-L-methionine (SAM) enzymes catalyze various free radical-mediated reactions. In these enzymes, the rate-determining SAM cleavage kinetically masks all the subsequent steps. Due to this kinetic masking, detailed mechanistic characterization of radical transformations catalyzed by these enzymes is very difficult. Here, we report a successful kinetic characterization of the radical C-C bond formation catalyzed by a MoaA radical SAM enzyme. MoaA catalyzes an unprecedented 3′,8-cyclization of GTP into 3′,8-cyclo-7,8-dihydro-GTP (3′,8-cH2GTP) during the molybdenum cofactor (Moco) biosynthesis. Through a series of EPR and biochemical characterization, we found that MoaA accumulates a 5′-deoxyadenos-4′-yl radical (5′-dA-C4′•) under the turnover conditions, and forms (4′S)-5′-deoxyadenosine ((4′S)-5′-dA), which is a C-4′ epimer of the naturally occurring (4′R)-5′-dA. Together with kinetic characterizations, these observations revealed the presence of a shunt pathway in which an on-pathway intermediate, GTP C-3′ radical, abstracts H-4′ atom from 5′-dA to transiently generate 5′-dA-C4′• that is subsequently reduced stereospecifically to yield (4′S)-5′-dA. Detailed kinetic characterization of the shunt and the main pathways provided the comprehensive view of MoaA kinetics, and determined the rate of the on-pathway 3′,8-cyclization step as 2.7 ± 0.7 s−1. Together with DFT calculations, this observation suggested that the 3′,8-cyclization is accelerated by 6 ∼ 9 orders of magnitude by MoaA. Potential contributions of the active-site amino acid residues, and their potential relationships with human Moco deficiency disease are discussed. This is the first determination of the magnitude of catalytic rate acceleration by a radical SAM enzyme, and provides the foundation for understanding how radical SAM enzymes achieve highly specific radical catalysis.

Aliphatic Ether Bond Formation Expands the Scope of Radical SAM Enzymes in Natural Product Biosynthesis

2019 ◽  
Vol 141 (27) ◽  
pp. 10610-10615 ◽  
Author(s):  
Kenzie A. Clark ◽  
Leah B. Bushin ◽  
Mohammad R. Seyedsayamdost
Keyword(s):  
Natural Product ◽  
Bond Formation ◽  
Radical Sam ◽  
Natural Product Biosynthesis ◽  
Radical Sam Enzymes ◽  
Ether Bond ◽  
Aliphatic Ether

scholarly journals The PqqD homologous domain of the radical SAM enzyme ThnB is required for thioether bond formation during thurincin H maturation

FEBS Letters ◽  
2015 ◽  
Vol 589 (15) ◽  
pp. 1802-1806 ◽  
Author(s):  
Beata M. Wieckowski ◽  
Julian D. Hegemann ◽  
Andreas Mielcarek ◽  
Linda Boss ◽  
Olaf Burghaus ◽  
...  
Keyword(s):  
Bond Formation ◽  
Radical Sam ◽  
Radical Sam Enzyme ◽  
Thurincin H

Two [4Fe-4S] Clusters Containing Radical SAM Enzyme SkfB Catalyze Thioether Bond Formation during the Maturation of the Sporulation Killing Factor

2013 ◽  
Vol 135 (3) ◽  
pp. 959-962 ◽  
Author(s):  
Leif Flühe ◽  
Olaf Burghaus ◽  
Beata M. Wieckowski ◽  
Tobias W. Giessen ◽  
Uwe Linne ◽  
...  
Keyword(s):  
Bond Formation ◽  
Radical Sam ◽  
Radical Sam Enzyme

scholarly journals Erratum: The radical SAM enzyme AlbA catalyzes thioether bond formation in subtilosin A

2012 ◽  
Vol 8 (8) ◽  
pp. 737-737 ◽  
Author(s):  
Leif Flühe ◽  
Thomas A Knappe ◽  
Michael J Gattner ◽  
Antje Schäfer ◽  
Olaf Burghaus ◽  
...  
Keyword(s):  
Bond Formation ◽  
Radical Sam ◽  
Radical Sam Enzyme

The radical SAM enzyme AlbA catalyzes thioether bond formation in subtilosin A

2012 ◽  
Vol 8 (4) ◽  
pp. 350-357 ◽  
Author(s):  
Leif Flühe ◽  
Thomas A Knappe ◽  
Michael J Gattner ◽  
Antje Schäfer ◽  
Olaf Burghaus ◽  
...  
Keyword(s):  
Bond Formation ◽  
Radical Sam ◽  
Radical Sam Enzyme

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

2020 ◽  
Vol 295 (49) ◽  
pp. 16665-16677
Author(s):  
Clémence Balty ◽  
Alain Guillot ◽  
Laura Fradale ◽  
Clémence Brewee ◽  
Benjamin Lefranc ◽  
...  
Keyword(s):  
Bond Formation ◽  
Large Family ◽  
Leader Peptide ◽  
Human Pathogens ◽  
Metabolic Potential ◽  
Radical Sam ◽  
Radical Sam Enzymes ◽  
Peptide Recognition ◽  
Recognition Element ◽  
Data Support

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 Cα-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-α-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 Cα 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 α,β-dehydro-amino acid intermediate during Cα-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.

scholarly journals 5-Deoxyadenosine Metabolism: More than “Waste Disposal”

2021 ◽  
pp. 1-12
Author(s):  
Johanna Rapp ◽  
Karl Forchhammer
Keyword(s):  
Current Knowledge ◽  
Shikimate Pathway ◽  
Central Carbon Metabolism ◽  
Competitive Growth ◽  
Radical Sam ◽  
Radical Sam Enzymes ◽  
Central Carbon ◽  
Domains Of Life ◽  
Radical Sam Enzyme ◽  
Salvage Pathways

5-Deoxyadenosine (5dAdo) is a by-product of many radical SAM enzyme reactions in all domains of life, and an inhibitor of the radical SAM enzymes themselves. Hence, pathways to recycle or dispose of this toxic by-product must exist but remain largely unexplored. In this review, we discuss the current knowledge about canonical and atypical 5dAdo salvage pathways that have been characterized in the last years. We highlight studies that report on how, in certain organisms, the salvage of 5dAdo via specific pathways can confer a growth advantage by providing either intermediates for the synthesis of secondary metabolites or a carbon source for the synthesis of metabolites of the central carbon metabolism. Yet, an alternative recycling route exists in organisms that use 5dAdo as a substrate to synthesize and excrete 7-deoxysedoheptulose, an allelopathic inhibitor of one enzyme of the shikimate pathway, thereby competing for their own niche. Remarkably, most steps of 5dAdo salvage are the result of the activity of promiscuous enzymes. This strategy enables even organisms with a small genome to synthesize bioactive compounds which they can deploy under certain conditions to gain a competitive growth advantage. We conclude emphasizing that, unexpectedly, 5dAdo salvage pathways seem not to be ubiquitously present, raising questions about the fate of such a toxic by-product in those species. This observation also suggests that additional 5dAdo salvage pathways, possibly relying on the activity of promiscuous enzymes, may exist. The future challenge will be to bring to light these “cryptic” 5dAdo recycling pathways.

Radical SAM Enzymes

2018 ◽  
Keyword(s):  
Radical Sam ◽  
Radical Sam Enzymes
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