At the confluence of ribosomally synthesized peptide modification and radical S-adenosylmethionine (SAM) enzymology

Abstract

Radical S-adenosylmethionine (RS) enzymology has emerged as a major biochemical strategy for the homolytic cleavage of unactivated C-H bonds. At the same time, the post-translational modification of ribosomally synthesized peptides is a rapidly expanding area of investigation. We discuss the functional cross-section of these two disciplines, highlighting the recently uncovered importance of protein-protein interactions, especially between the peptide substrate and its chaperone, which functions either as a stand-alone protein or as an N-terminal fusion to the respective RS enzyme. The need for further work on this class of enzymes is emphasized, given the poorly understood roles performed by multiple, auxiliary iron-sulfur clusters and the paucity of protein X-ray structural data.Modern biochemistry has seen the exponential growth of two exciting areas of research that focus individually on the post-translational modification of ribosomally synthesized and post-translationally modified peptides (RiPPs) 2 (1) and on the structure and mechanism of free radical conversions catalyzed by radical S-adenosylmethionine (RS) enzymes (2, 3). These separate fields have recently converged within a growing family of enzymes that bring about free radical-based, post-translational modifications on ribosomally produced peptide substrates. RS enzymes function by cleavage of a [4Fe-4S] clusterbound S-adenosylmethionine to form methionine and a deoxyadenosyl radical. This radical then initiates the reaction by abstracting a hydrogen atom from the substrate. The [4Fe-4S] cluster that accomplishes this reaction has a characteristic CX 3 CXC motif, with each of the cysteines coordinated to a single iron, leaving an open coordination sphere where SAM binds and is cleaved. A subset of RS enzymes belongs to a family that contains an additional conserved structural motif annotated as a SPASM domain and referred to as RS-SPASM proteins. Although the exact function of the SPASM domain is unknown, it has been shown that it houses generally two additional iron-sulfur clusters that are critical in RS-SPASM chemistry. Using the perspective of the pathway for production of the bacterial cofactor pyrroloquinoline quinone (PQQ), we compare and highlight some of the unique properties among these RS-SPASM-dependent RiPP systems. Bioinformatics analyses suggest that the family members will extend far beyond the examples presented herein.

Iron catalysis of lipid peroxidation in ferroptosis: Regulated enzymatic or random free radical reaction?

“…The role of reduced iron as the electron donor in many reactions, including photosynthesis, and oxidized iron as the universal terminal electron acceptor in respiratory chains, as well as the catalytic propensities of iron as a key cofactor for a variety of enzymatic reactions determined its irreplaceable functions in all living organisms [3][4][5][6][7]. The newly emerging field of radical enzymology points to a possibly important role of iron in primordial hydrogen abstraction reactions occurring anaerobically in primitive organism through tightly coordinated alkyl radical driven mechanisms [8]. The essential high chemical reactivity of iron also imposed the creation of highly specialized, complex mechanisms of competition for iron between host cells and invading bacterial pathogens [1], along with those for transport and control of extra-and intra-cellular iron, based on tight binding of this metal by specialized proteins.…”

Methods for Expression, Purification, and Characterization of PqqE, a Radical SAM Enzyme in the PQQ Biosynthetic Pathway

“…Despite the significant importance of PQQ to human health, the details of the biosynthetic pathway of this molecule have been, until recently, largely unknown. Recent studies by our group revealed that a key step in the PQQ biosynthesis involves the formation of a C–C bond between two amino acids in the peptide substrate PqqA, catalyzed by the radical SAM enzyme PqqE in complex with the chaperone PqqD (Barr et al, 2016; Latham, Barr, & Klinman, 2017; Latham et al, 2015). This reaction is both complicated and fascinating not only because it is an O 2 -sensitive reaction occurring in an aerobic bacteria ( Mex ) but also due to the fact that it requires precise redox control and interaction among multiprotein complexes.…”

Occurrence, function, and biosynthesis of mycofactocin

“…It was observed that MftC belongs to a RS enzyme subfamily that contains an elongated C-terminal domain annotated as a SPASM domain (Subtilosin A, Pyrroloquinoline quinone, Anaerobic Sulfatase maturating enzyme, and Mycofactocin) (Haft and Basu 2011). The proteins associated with the RS-SPASM subfamily are known peptide modifiers that have been shown to catalyze intramolecular C-S bonds and C-C bonds and oxidative decarboxylation reactions on the precursor peptide (Flühe et al 2012;Flühe et al 2013;Wieckowski et al 2015;Barr et al 2016;Khaliullin et al 2016;Khaliullin et al 2017a;Latham et al 2017a;Schramma and Seyedsayamdost 2017). Using the MftC clade as a starting point, Haft identified the remaining five mft genes in the immediate neighborhood localized around mftC (Figure 1).…”

“…In both cases, the first step requires the RS enzymes PqqE and MftC, respectively. Both enzymes catalyze carbon-carbon bond formation between an sp 2 and an sp 3 hybridized carbon on their precursor peptides, in the presence of an accessory RRE domain (PqqD and MftB, respectively), resulting in a chemically crosslinked peptide (Latham et al 2015;Barr et al 2016;Khaliullin et al 2016;Khaliullin et al 2017a;Latham et al 2017a). However, while PqqE catalyzes the carboncarbon bond formation between glutamate and tyrosine on its precursor peptide PqqA in a single step (Figure 3), MftC catalyzes a two-step modification of the precursor peptide MftA (Figure 4) (Barr et al 2016;Khaliullin et al 2017a).…”

“…MftA is a small ribosomally synthesized peptide that contains a strictly conserved Cterminal sequence -IDGXCGVY. In addition, the cluster encodes for MftB, a small protein with predicted structural homology to the RiPP recognition element (RRE) PqqD of the PQQ biosynthetic pathway (Latham et al 2017a). The protein MftC is a putative radical Sadenosylmethionine (RS) enzyme with low sequence homology to the PQQ biosynthetic enzyme PqqE (Haft and Basu 2011).…”