Journey on the Radical SAM Road as an Accidental Pilgrim

Abstract

Radical S-adenosyl-l-methionine (SAM) enzymes catalyze a diverse group of complex transformations in all aspects of cellular physiology. These metalloenzymes bind SAM to a 4Fe-4S cluster and reductively cleave SAM to generate a 5'-deoxyadenosyl radical, which generally initiates the catalytic cycle by catalyzing a H atom to activate the substrate for subsequent chemistry. This perspective will focus on our discovery of several members of this superfamily of enzymes, with a particular emphasis on the current state of the field, challenges, and outlook.

Figure 1

Institute for Enzyme Research at the University of Wisconsin—Madison. The Frey and Reed laboratories were located on the 5th and 4th floors. The photo from Cory Coyle/CC-BY-SA-3.0 was used without modification.

Figure 2

Biosynthesis of 7-deazapurines. GTP cyclohydrolase I (GCH I), CPH₄ synthase (QueD), and 7-carboxy-7-deazaguanine (CDG) synthase catalyze the conversion of GTP to CDG, which is a common precursor to 7-deazapurines. QueG catalyzes the B₁₂-dependent reduction of oQ-tRNA to the Q-tRNA.

Figure 3

CDG synthase catalyzes a radical-mediated ring contraction reaction.

Figure 4

Structure of Burkholderia multivorans CDG synthase bound to substrate (CPH₄) or product (CDG). (A) CDG synthase is a dimer of identical subunits. The yellow/orange spheres in the structure denote the iron–sulfur cluster, and the green sphere is the required magnesium divalent cation. The substrate (B) and product (C) bind similarly and make identical interactions with a small number of residues. The separation between C-5′ of SAM and C-6 hydrogen of the substrate is shown by the dashed line. The H was modeled with the PyMol Molecular Graphics System (version 2.0 Schrödinger, LLC).

Figure 5

Divergent fates of 6-CP incubated with CDG synthase. In the absence of reductant, the analogue is adenosylated by SAM to form a C–O bond. In the presence of reductant, radical addition followed by oxidative decarboxylation leads to a C–C linked dAdo adduct.

Figure 6

Biosynthesis of wybutosine. The biosynthetic pathway to yW consists of five SAM-dependent steps. TYW1 catalyzes the second step, which entails condensation of pyruvate with m¹G to form imG-14. The reaction product retains C-2 and C-3 of pyruvate. The fate of C-1 of pyruvate remains to be established.

Figure 7

Mechanism of TYW1. The enzyme binds pyruvate as a Schiff base to a conserved Lys residue in the active site. The substrate is activated for radical addition by H atom transfer from m¹G to dAdo•. Radical addition is followed by C–C bond cleavage to release C-1 of the pyruvate as a yet unknown product. Transimination and aromatization complete the catalytic cycle.

Figure 8

Reaction catalyzed by SbtM. The reaction catalyzed by SbtM is hypothesized to occur via oxidation of the Cys/SeCys side chain, followed by ring formation and an additional oxidation step to form the product. Each of the oxidation steps are thought to utilize SAM and involve H atom transfer from the β-carbon of the side chain to dAdo•. A thio/selenoaldehyde-like intermediate is proposed on the pathway to the final product.