Eukaryotic TYW1 Is a Radical SAM Flavoenzyme

Abstract

TYW1 is a radical S-adenosyl-L-methionine (SAM) enzyme that catalyzes the condensation of pyruvate and Nmethylguanosine-containing tRNA Phe , forming 4-demethylwyosine-containing tRNA Phe . Homologues of TYW1 are found in both archaea and eukarya; archaeal homologues consist of a single domain, while eukaryal homologues contain a flavin binding domain in addition to the radical SAM domain shared with archaeal homologues. In this study, TYW1 from Saccharomyces cerevisiae (ScTYW1) was heterologously expressed in Escherichia coli and purified to homogeneity. ScTYW1 is purified with 0.54 ± 0.07 and 4.2 ± 1.9 equiv of flavin mononucleotide (FMN) and iron, respectively, per mole of protein, suggesting the protein is ∼50% replete with Fe−S clusters and FMN. While both NADPH and NADH are sufficient for activity, significantly more product is observed when used in combination with flavin nucleotides. ScTYW1 is the first example of a radical SAM flavoenzyme that is active with NAD(P)H alone.

Lysine 2,3-Aminomutase and a Newly Discovered Glutamate 2,3-Aminomutase Produce β-Amino Acids Involved in Salt Tolerance in Methanogenic Archaea

“…Many radical SAM enzymes with C-terminal SPASM or twitch domains contain auxiliary Fe−S clusters with proposed roles in electron transfer, 60,61 and a recent study has characterized a eukaryotic radical SAM enzyme that is fused to a flavodoxin domain. 62 Analysis of the extended N-terminus of MmarC7_1783 revealed only three His residues and one Cys residue that are not highly conserved or within close proximity to one another; thus, this domain likely does not play a role in metal binding. Further sequence and structural analyses did not show any similarities to known cofactorbinding domains, and BLAST searches indicate that there are no other proteins with a similar domain besides other putative long-aminomutases in various microorganisms.…”

“…We first considered that this domain may bind a redox cofactor involved in shuttling an electron to the radical SAM [4Fe–4S] cluster. Many radical SAM enzymes with C-terminal SPASM or twitch domains contain auxiliary Fe–S clusters with proposed roles in electron transfer, , and a recent study has characterized a eukaryotic radical SAM enzyme that is fused to a flavodoxin domain . Analysis of the extended N-terminus of MmarC7_1783 revealed only three His residues and one Cys residue that are not highly conserved or within close proximity to one another; thus, this domain likely does not play a role in metal binding.…”

Journey on the Radical SAM Road as an Accidental Pilgrim

“…The breakthrough was when we introduced a second plasmid encoding the suf operon in the expression strains. This led to the purification of the Saccharomyces cerevisiae TYW1 ( Sc TYW1), which is now the first characterized radical SAM flavoenzyme . After initially establishing that the protein carries out the same reaction as the archaeal homologue, we focused on the role of the flavodoxin domain.…”

“…This led to the purification of the Saccharomyces cerevisiae TYW1 (ScTYW1), which is now the first characterized radical SAM flavoenzyme. 39 After initially establishing that the protein carries out the same reaction as the archaeal homologue, we focused on the role of the flavodoxin domain. Remarkably, the formation of imG-14 with ScTYW1 requires only the presence of NAD(P)H, pyruvate, and m 1 G-containing substrate.…”

Insertion of 4-Demethylwyosine in tRNAPhe Catalyzed by the Radical S-Adenosyl-l-methionine Enzyme TYW1 Entails Oxidative Cleavage of Pyruvate to Form CO2

“…14−17 All TYW1 homologues contain two 4Fe-4S clusters, while the eukaryal homologue is unique among RS enzymes and binds an equivalent of flavin mononucleotide. 18 The two 4Fe-4S clusters in TYW1 are hypothesized to bind two different substrates. One of the 4Fe-4S clusters, bound by the characteristic CxxxCxxC motif, binds and reductively cleaves SAM, which is typical of RS enzymes.…”