Radical SAM


Radical SAM is a designation for a superfamily of enzymes that use a 4Fe-4S|+ cluster to reductively cleave S-adenosyl-L-methionine to generate a radical, usually a 5′-deoxyadenosyl radical, as a critical intermediate. These enzymes utilize this potent radical intermediate to perform an array of unusual transformations, often to functionalize unactivated C-H bonds. Radical SAM enzymes are involved in cofactor biosynthesis, enzyme activation, peptide modification, post-transcriptional and post-translational modifications, metalloprotein cluster formation, tRNA modification, lipid metabolism, biosynthesis of antibiotics and natural products etc. The vast majority of known radical SAM enzymes belong to the radical SAM superfamily, and have a cysteine-rich motif that matches or resembles CxxxCxxC.

History and mechanism

As of 2001, 645 unique radical SAM enzymes have been identified from 126 species in all three domains of life. According to the EFI and SFLD databases, more than 220,000 radical SAM enzymes are predicted to be involved in 85 types of biochemical transformations.
The mechanism for these reactions entail transfer of a methyl or adenosyl group from sulfur to iron. The resulting organoiron complex subsequently releases the organic radical. The latter step is reminiscent of the behavior of adenosyl and methyl cobalamins.

Nomenclature

All enzymes including radical SAM superfamily follow an easy guideline for systematic naming. Systematic naming of enzymes allows a uniform naming process that is recognized by all scientists to understand corresponding function. The first word of the enzyme name often shows the substrate of the enzyme. The position of the reaction on the substrate will also be in the beginning portion of the name. Lastly, the class of the enzyme will be described in the other half of the name which will end in suffix -ase. The class of an enzyme will describe what the enzyme is doing or changing on the substrate. For example, a ligase combines two molecules to form a new bond.
and is responsible for radical generation. β-sheets are colored yellow and α-helices are shown in cyan. |alt=

Reaction classification

Representative/Prototype enzymes will only be mentioned for each reaction scheme. The audience is highly encouraged to research more into current studies on radical SAM enzymes. Many of which are responsible for fascinating yet important reactions.
Radical SAM enzymes and their mechanisms known before 2008 are well-summarized by Frey et al, 2008. Since 2015, more review articles on radical SAM enzymes are open to the public. The following are only a few out of many informative resources on radical SAM enzymes.
  1. Recent Advances in Radical SAM Enzymology: New Structures and Mechanisms:
  2. Radical S-Adenosylmethionine Enzymes:
  3. Radical S-Adenosylmethionine Enzymes in Cofactor Biosynthesis: A Treasure Trove of Complex Organic Radical Rearrangement Reactions:
  4. Molecular architectures and functions of radical enzymes and their activating proteins:

    Carbon methylation

Radical SAM methylases/methyltransferases are one of the largest yet diverse subgroups and are capable of methylating a broad range of unreactive carbon and phosphorus centers. These enzymes are divided into four classes with representative methylation mechanisms. The shared characteristic of the three major classes A, B and C is the usage of SAM, split into two distinct roles: one as a source of a methyl group donor, and the second as a source of 5'-dAdo radical. The recently documented class D utilizes a different methylation mechanism.

Class A sub-family

Methythiotransferases belong to a subset of radical SAM enzymes that contain two + clusters and one radical SAM domain. Methylthiotransferases play a major role in catalyzing methylthiolation on tRNA nucleotides or anticodons through a redox mechanism. Thiolation modification is believed to maintain translational efficiency and fidelity.
MiaB and RimO are both well-characterized and bacterial prototypes for tRNA-modifying methylthiotransferases
eMtaB is the designated methylthiotransferase in eukaryotic and archaeal cells. eMtaB catalyzes the methylthiolation of tRNA at position 37 on N6-threonylcarbamoyladenosine. A bacterial homologue of eMtaB, YqeV has been reported and suggested to function similarly to MiaB and RimO.

Sulfur insertion into unreactive C-H bonds

Sulfurtransferases are a small subset of radical SAM enzymes. Two well-known examples are BioB and LipA which are independently responsible for biotin synthesis and lipoic acid metabolism, respectively.
is a metallozyme with essential function in the biological nitrogen fixation reaction. The M-cluster and P-cluster are highly unique metalloclusters present in nitrogenase. The best-studied nitrogenase up-to-date is Mo nitrogenase with M-cluster and P-cluster bearing important roles in substrate reduction. The active site of Mo nitrogenase is the M-cluster, a metal-sulfur cluster containing a carbide at its core. Within the biosynthesis of M-cluster, radical SAM enzyme NifB has been recognized to catalyze a carbon insertion reaction, leading to formation of a Mo/homocitrate-free precursor of M-cluster.

Anaerobic oxidative decarboxylation

Glycyl radical enzyme activating enzymes are radical SAM subset that can house a stable and catalytically essential glycyl radical in their active state. The underlying chemistry is considered to be the simplest in the radical SAM superfamily with H-atom abstraction by the 5'-dAdo radical being the product of the reaction. A few examples include:
Radical SAM enzymes that can catalyze sulfur-to-alpha carbon or sulfur-to-beta thioether cross-linked peptides are important to generate an essential class of peptide with significant antibacterial, spermicidal and hemolytic properties. Another common name for this peptide class is ribosomally synthesized and post-translationally modified peptides.
Another important subset of peptide-modifying radical SAM enzymes is SPASM/Twitch domain-carrying enzymes. SPASM/Twitch enzymes carry a functionalized C-terminal extension for the binding of two clusters, especially important in post-translational modifications of peptides.
The following examples are representative enzymes that can catalyze peptide modifications to generate specific natural products or cofactors.
  1. TsrM in thiostrepton biosynthesis
  2. PoyD and PoyC in polytheonamide biosynthesis
  3. TbtI in thiomuracin biosynthesis
  4. NosN in nosiheptide biosynthesis
  5. MoaA in molybdopterin biosynthesis
  6. PqqE in pyrroloquinoline quinone biosynthesis
  7. TunB in tunicamycin biosynthesis
  8. OxsB in oxetanocin biosynthesis
  9. BchE in anaerobic bacteriochlorophyll biosynthesis
  10. F0 synthases in F420 cofactor biosynthesis
  11. MqnE and MqnC in menaquinone biosynthesis
  12. QhpD in post-translational processing of quinohemoprotein amine dehydrogenase

    Epimerization

Radical SAM epimerases are responsible for the regioselective introduction of D-amino acids into RiPPs. Two well-known enzymes have been thoroughly described in RiPP biosynthetic pathways.
Another subset of radical SAM superfamily has been shown to catalyze carbon skeleton rearrangements especially in the areas of DNA repair and cofactor biosynthesis.
Microbes have been extensively used for the discovery of new antibiotics. However, a growing public concern of multi-drug resistant pathogens has been emerging in the last few decades. Thus, newly developed or novel antibiotics are in utmost demand. Ribosomally synthesized and post-translationally modified peptides are getting more attention as a newer and major group of antibiotics thanks to having a very narrow of activity spectrum, which can benefit patients, as their side effects will be lesser than the broad-spectrum antibiotics. Below are a few examples of radical SAM enzymes have been shown to be promising targets for antibiotic and antiviral development.

Radical

Examples of radical SAM enzymes found within the radical SAM superfamily include:
In addition, several non-canonical radical SAM enzymes have been described. These cannot be recognized by the Pfam hidden Markov model PF04055, but still use three Cys residues as ligands to a 4Fe4S cluster and produce a radical from S-adenosylmethionine. These include