Kaitlin S. Duschene

Radical S-Adenosylmethionine Enzymes

It was once widely held that nearly all reactions in biology were catalyzed via mechanisms involving paired electron species. Beginning approximately 40 years ago, this paradigm was repeatedly challenged as examples of enzymatic reactions involving organic radical intermediates began to emerge, and it is now well accepted that biochemical reactions often involve organic radicals. Indeed, some of the most intensely studied metalloenzymes, including cytochrome P450, methane monooxygenase, ribonucleotide reductase, and the adenosylcobalamin (B12) enzymes, catalyze reactions employing organic radical intermediates. As a general rule, enzymes utilizing radical mechanisms catalyze reactions that would be difficult or impossible to catalyze by polar mechanisms, most often involving H-atom abstraction from an unactivated C–H bond. Among the more recent additions to the enzymes that catalyze radical reactions are the radical S-adenosylmethionine (radical SAM) enzymes, which were first classified as a superfamily in 2001.1 These enzymes utilize a [4Fe–4S] cluster and SAM to initiate a diverse set of radical reactions, in most or all cases via generation of a 5′-deoxyadenosyl radical (dAdo•) intermediate. Although 2001 marked the identification of this superfamily largely through bioinformatics, the discovery of iron metalloenzymes utilizing SAM to initiate radical reactions precedes this date by more than a decade. For example, early studies on the activation of pyruvate formate-lyase showed that it involved the generation of a stable protein radical,2 and was stimulated by the presence of iron, SAM, and an “activating component” from the cell extract now known to be the pyruvate-formate lyase activating enzyme (PFL-AE).3 The radical on PFL was ultimately shown to be located on a specific glycine residue,4 and was one of the first stable protein radicals characterized. PFL-AE was ultimately shown to contain a catalytically essential iron–sulfur cluster,5 and to use SAM as an essential component of PFL activation.6 The anaerobic ribonucleotide reductase, similar to PFL, contains a stable glycyl radical that was shown in early work to require an iron–sulfur cluster and SAM for activation.7 Likewise, preliminary investigations on lysine 2,3-aminomutase (LAM) published in 1970 demonstrated activation by ferrous ion and a strict requirement for SAM.8 Like PFL-AE, LAM was ultimately found to contain a catalytically essential iron–sulfur cluster.9 Work in Perry Frey’s lab showed that LAM used the adenosyl moiety of SAM to mediate hydrogen transfer in a manner similar to adenosylcobalamin-dependent rearrangements, implicating radical intermediates.10 Biotin synthase was first reported to require iron and SAM in 1995,11 and was subsequently shown to contain iron–sulfur clusters and to catalyze a radical reaction.12 These four enzyme systems (PFL/PFL-AE, aRNR, LAM, and biotin synthase) provided early indications of a new type of biological cofactor consisting of an iron–sulfur cluster and SAM, which initiate radical reactions using a fundamental new mechanism of catalysis.13 What none of us in the field in the early days probably anticipated, however, was just how ubiquitous these enzymes would turn out to be. The initial report of the superfamily by Sofia et al. identified ∼600 members;1 however, now that number is ∼48 100 members.14 These enzymes are found across the phylogenetic kingdom and catalyze an amazingly diverse set of reactions, the vast majority of which have yet to be characterized. This Review will begin by summarizing unifying features of radical SAM enzymes, and in subsequent sections delve further into the biochemical, spectroscopic, structural, and mechanistic details for those enzymes that have been characterized. In most cases, these enzymes are grouped by reaction type; however, in two cases (syntheses of modified tetrapyrroles and complex metal cluster cofactors), we have chosen to group together several radical SAM enzymes that catalyze different reaction types but which act together in the same or related metabolic pathways.

HydF as a scaffold protein in [FeFe] hydrogenase H‐cluster biosynthesis

In an effort to determine the specific protein component(s) responsible for in vitro activation of the [FeFe] hydrogenase (HydA), the individual maturation proteins HydE, HydF, and HydG from Clostridium acetobutylicum were purified from heterologous expressions in Escherichia coli. Our results demonstrate that HydF isolated from a strain expressing all three maturation proteins is sufficient to confer hydrogenase activity to purified inactive heterologously expressed HydA (expressed in the absence of HydE, HydF, and HydG). These results represent the first in vitro maturation of [FeFe] hydrogenase with purified proteins, and suggest that HydF functions as a scaffold upon which an H‐cluster intermediate is synthesized.

The antiviral protein viperin is a radical SAM enzyme

Viperin, an interferon‐inducible antiviral protein, is shown to bind an iron‐sulfur cluster, based on iron analysis as well as UV–Vis and electron paramagnetic resonance spectroscopic data. The reduced protein contains a [4Fe‐4S]1+ cluster whose g‐values are altered upon addition of S‐adenosylmethionine (SAM), consistent with SAM coordination to the cluster. Incubation of reduced viperin with SAM results in reductive cleavage of SAM to produce 5′‐deoxyadenosine (5′‐dAdo), a reaction characteristic of the radical SAM superfamily. The 5′‐dAdo cleavage product was identified by a combination of HPLC and mass spectrometry analysis.

Control of radical chemistry in the AdoMet radical enzymes.

The radical AdoMet superfamily comprises a diverse set of >2800 enzymes that utilize iron-sulfur clusters and S-adenosylmethionine (SAM or AdoMet) to initiate a diverse set of radical-mediated reactions. The intricate control these enzymes exercise over the radical transformations they catalyze is an amazing feat of elegance and sophistication in biochemistry. This review focuses on the accumulating evidence for how these enzymes control this remarkable chemistry, including controlling the reactivity between the iron-sulfur cluster and AdoMet, and controlling the subsequent radical transformations.

Biochemical and Kinetic Characterization of Radical S-Adenosyl-l-methionine Enzyme HydG

The radical S-adenosyl-L-methionine (AdoMet) enzyme HydG is one of three maturase enzymes involved in [FeFe]-hydrogenase H-cluster assembly. It catalyzes L-tyrosine cleavage to yield the H-cluster cyanide and carbon monoxide ligands as well as p-cresol. Clostridium acetobutylicum HydG contains the conserved CX3CX2C motif coordinating the AdoMet binding [4Fe-4S] cluster and a C-terminal CX2CX22C motif proposed to coordinate a second [4Fe-4S] cluster. To improve our understanding of the roles of each of these iron-sulfur clusters in catalysis, we have generated HydG variants lacking either the N- or C-terminal cluster and examined these using spectroscopic and kinetic methods. We have used iron analyses, UV-visible spectroscopy, and electron paramagnetic resonance (EPR) spectroscopy of an N-terminal C96/100/103A triple HydG mutant that cannot coordinate the radical AdoMet cluster to unambiguously show that the C-terminal cysteine motif coordinates an auxiliary [4Fe-4S] cluster. Spectroscopic comparison with a C-terminally truncated HydG (ΔCTD) harboring only the N-terminal cluster demonstrates that both clusters have similar UV-visible and EPR spectral properties, but that AdoMet binding and cleavage occur only at the N-terminal radical AdoMet cluster. To elucidate which steps in the catalytic cycle of HydG require the auxiliary [4Fe-4S] cluster, we compared the Michaelis-Menten constants for AdoMet and L-tyrosine for reconstituted wild-type, C386S, and ΔCTD HydG and demonstrate that these C-terminal modifications do not affect the affinity for AdoMet but that the affinity for L-tyrosine is drastically reduced compared to that of wild-type HydG. Further detailed kinetic characterization of these HydG mutants demonstrates that the C-terminal cluster and residues are not essential for L-tyrosine cleavage to p-cresol but are necessary for conversion of a tyrosine-derived intermediate to cyanide and CO.

H-Cluster assembly during maturation of the [FeFe]-hydrogenase

The organometallic H-cluster at the active site of the [FeFe]-hydrogenase serves as the site of reversible binding and reduction of protons to produce H2. The H-cluster is unique in biology, and consists of a 2Fe subcluster tethered to a typical [4Fe–4S] cluster by a single cysteine ligand. The remaining ligands to the 2Fe subcluster include three carbon monoxides, two cyanides, and a dithiomethylamine. This mini-review will focus on the significant advances in recent years in understanding the pathway for H-cluster biosynthesis, as well as the structures, roles, and mechanisms of the three enzymes directly involved.

Radical SAM catalysis via an organometallic intermediate with an Fe–[5′-C]-deoxyadenosyl bond

Catching a radical in action Many enzymes catalyze reactions through the production of radical intermediates. Radical SAM enzymes, the largest superfamily of enzymes in nature, do this by using an iron-sulfur cluster to cleave S-adenosylmethionine and produce a radical intermediate. Using freeze quenching, Horitani et al. were able to trap a previously unseen radical intermediate from bacterial pyruvate formate-lyase activating enzyme. Spectroscopy revealed that the intermediate consists of a short-lived covalent bond between the terminal carbon of 5′-deoxyadenosyl and the single iron atom of the iron-sulfur cluster. Not only does the observation of this radical expand our mechanistic understanding of radical SAM enzymes, but it expands the range of enzyme active sites or cofactors that function through an organometallic center. Science, this issue p. 822 Freeze-quench experiments trap a radical intermediate on pyruvate formate-lyase activating enzyme. Radical S-adenosylmethionine (SAM) enzymes use a [4Fe-4S] cluster to cleave SAM to initiate diverse radical reactions. These reactions are thought to involve the 5′-deoxyadenosyl radical intermediate, which has not yet been detected. We used rapid freeze-quenching to trap a catalytically competent intermediate in the reaction catalyzed by the radical SAM enzyme pyruvate formate-lyase activating enzyme. Characterization of the intermediate by electron paramagnetic resonance and 13C, 57Fe electron nuclear double-resonance spectroscopies reveals that it contains an organometallic center in which the 5′ carbon of a SAM-derived deoxyadenosyl moiety forms a bond with the unique iron site of the [4Fe-4S] cluster. Discovery of this intermediate extends the list of enzymatic bioorganometallic centers to the radical SAM enzymes, the largest enzyme superfamily known, and reveals intriguing parallels to B12 radical enzymes.

Viperin: a radical response to viral infection

Abstract One of the first lines of defense of the host immune response to infection is upregulation of interferons, which play a vital role in triggering the early nonspecific antiviral state of the host. Interferons prompt the generation of numerous downstream products, known as interferon-stimulated genes (ISGs). One such ISG found to be either directly induced by type I, II, and III interferons or indirectly through viral infection is the ‘virus inhibitory protein, endoplasmic reticulum-associated, interferon-inducible’ protein, or viperin. Not only is viperin capable of combating a wide array of viral infections but its upregulation is also observed in the presence of endotoxins, various bacterial infections, or even in response to other immune stimuli, such as atherosclerotic lesions. Recent advances in the understanding of possible mechanisms of action of viperin involve, but are perhaps not limited to, interaction with farnesyl pyrophosphate synthase and disruption of lipid raft domains to prevent viral bud release, inhibition of hepatitis C virus secretory proteins, and coordination to lipid droplets and inhibition of viral replication. Unexpectedly, new insight into the human cytomegalovirus induction of this antiviral protein demonstrates that mitochondrial viperin plays a necessary and beneficial role for viral propagation.

Paradigm Shift for Radical S-Adenosyl-l-methionine Reactions: The Organometallic Intermediate Ω Is Central to Catalysis

Radical S-adenosyl-l-methionine (SAM) enzymes comprise a vast superfamily catalyzing diverse reactions essential to all life through homolytic SAM cleavage to liberate the highly reactive 5′-deoxyadenosyl radical (5′-dAdo·). Our recent observation of a catalytically competent organometallic intermediate Ω that forms during reaction of the radical SAM (RS) enzyme pyruvate formate-lyase activating-enzyme (PFL-AE) was therefore quite surprising, and led to the question of its broad relevance in the superfamily. We now show that Ω in PFL-AE forms as an intermediate under a variety of mixing order conditions, suggesting it is central to catalysis in this enzyme. We further demonstrate that Ω forms in a suite of RS enzymes chosen to span the totality of superfamily reaction types, implicating Ω as essential in catalysis across the RS superfamily. Finally, EPR and electron nuclear double resonance spectroscopy establish that Ω involves an Fe–C5′ bond between 5′-dAdo· and the [4Fe–4S] cluster. An analogous organometallic bond is found in the well-known adenosylcobalamin (coenzyme B12) cofactor used to initiate radical reactions via a 5′-dAdo· intermediate. Liberation of a reactive 5′-dAdo· intermediate via homolytic metal–carbon bond cleavage thus appears to be similar for Ω and coenzyme B12. However, coenzyme B12 is involved in enzymes catalyzing only a small number (∼12) of distinct reactions, whereas the RS superfamily has more than 100 000 distinct sequences and over 80 reaction types characterized to date. The appearance of Ω across the RS superfamily therefore dramatically enlarges the sphere of bio-organometallic chemistry in Nature.