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.

Keywords: RiPPs; SPASM; iron–sulfur protein; oxidation–reduction (redox); peptide biosynthesis; post-translational modification (PTM); radical; radical SAM.

Figure 1.

PQQ biosynthetic pathway. The ribosomally-produced peptide PqqA, containing a leader sequence and the to-be-modified Glu and Tyr, is recognized and bound by the peptide chaperone PqqD (1). The complex associates with PqqE, along with one equivalent of SAM (2). The radical SAM Fe-S cluster is reduced, and this electron is then transferred to reductively cleave SAM (3 and 4). The newly-produced deoxyadenosyl radical abstracts hydrogen from the glutamate γ-carbon, leading to formation of a carbon–carbon bond between two residues and a radical on the tyrosine (5). Oxidation of the tyrosyl radical leads to the cross-linked peptide product, which is later hydrolyzed and oxidized to give AHQQ, the substrate for PqqC, which converts AHQQ to PQQ in an oxygen-dependent manner.

Figure 2.

A, X-ray structure of the canonical AnSME from C. perfringens (PDB code 4K36) indicates the presence of a SPASM domain (red) in addition to the conserved TIM barrel (gray). The TIM barrel contains the RS [4Fe-4S] cluster (black asterisk), ligated by three cysteines and SAM (green), whereas the SPASM domain contains an auxiliary I cluster (blue asterisk) and an auxiliary II cluster (red asterisk), both ligated by four cysteines. B, chemical reaction catalyzed by AnSME.

Figure 3.

A, X. campestris PqqD crystal structure (PDB code 3G2B) determined the oligomeric state to be a dimer. B, solution NMR ¹H,¹⁵N-HSQC experiments on the monomeric M. extorquens AM1 PqqD (PDB code 5SXY) identify residues with significant chemical shifts when bound to PqqA (violet residues) and PqqE (green residues). C, homology-based modeling predicts that the putative peptide chaperone domains of MftB (light blue), QhpD (blue), AlbA (cyan), ThnB (violet), StrB (partial, light gray), and SCIFF maturase (magenta) all have similar structures to that of PqqD (dark gray). D, overlay of the pathway peptides from NisB (light blue) and LynD (dark blue) onto the structure for PqqD, where purple represents residues that are altered in the PqqD–PqqA complex, and green represents positions altered in the ternary complex consisting of PqqD, PqqA, and PqqE. E, complex of MccB with its cognate substrate where the peptide is colored purple.