Enzymes of the radicalS-adenosyl-l-methionine (radical SAM, RS) superfamily, the largest in nature, catalyze remarkably diverse reactions initiated by H-atom abstraction. Glycyl radical enzyme activating enzymes (GRE-AEs) are a growing class of RS enzymes that generate the catalytically essential glycyl radical of GREs, which in turn catalyze essential reactions in anaerobic metabolism. Here, we probe the reaction of the GRE-AE pyruvate formate-lyase activating enzyme (PFL-AE) with the peptide substrate RVSG734YAV, which mimics the site of glycyl radical formation on the native substrate, pyruvate formate-lyase. Time-resolved freeze-quench electron paramagnetic resonance spectroscopy shows that at short mixing times reduced PFL-AE + SAM reacts with RVSG734YAV to form the central organometallic intermediate, Ω, in which the adenosyl 5′C is covalently bound to the unique iron of the [4Fe–4S] cluster. Freeze-trapping the reaction at longer times reveals the formation of the peptide G734• glycyl radical product. Of central importance, freeze-quenching at intermediate times reveals that the conversion of Ω to peptide glycyl radical is not concerted. Instead, homolysis of the Ω Fe–C5′ bond generates the nominally “free” 5′-dAdo• radical, which is captured here by freeze-trapping. During cryoannealing at 77 K, the 5′-dAdo• directly abstracts an H-atom from the peptide to generate the G734• peptide radical trapped in the PFL-AE active site. These observations reveal the 5′-dAdo• radical to be a well-defined intermediate, caught in the act of substrate H-atom abstraction, providing new insights into the mechanistic steps of radical initiation by RS enzymes. 
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                            Highlighting the Unique Roles of Radical S -Adenosylmethionine Enzymes in Methanogenic Archaea
                        
                    
    
            ABSTRACT Radical S -adenosylmethionine (SAM) enzymes catalyze an impressive variety of difficult biochemical reactions in various pathways across all domains of life. These metalloenzymes employ a reduced [4Fe-4S] cluster and SAM to generate a highly reactive 5′-deoxyadenosyl radical that is capable of initiating catalysis on otherwise unreactive substrates. Interestingly, the genomes of methanogenic archaea encode many unique radical SAM enzymes with underexplored or completely unknown functions. These organisms are responsible for the yearly production of nearly 1 billion tons of methane, a potent greenhouse gas as well as a valuable energy source. Thus, understanding the details of methanogenic metabolism and elucidating the functions of essential enzymes in these organisms can provide insights into strategies to decrease greenhouse gas emissions as well as inform advances in bioenergy production processes. This minireview provides an overview of the current state of the field regarding the functions of radical SAM enzymes in methanogens and discusses gaps in knowledge that should be addressed. 
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                            - Award ID(s):
- 2105598
- PAR ID:
- 10411591
- Editor(s):
- Maupin-Furlow, Julie A.
- Date Published:
- Journal Name:
- Journal of Bacteriology
- Volume:
- 204
- Issue:
- 8
- ISSN:
- 0021-9193
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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