Eukaryotic TYW1 Is a Radical SAM Flavoenzyme.

TYW1 is a radical -adenosyl-l-methionine (SAM) enzyme that catalyzes the condensation of pyruvate and -methylguanosine-containing tRNA, forming 4-demethylwyosine-containing tRNA. 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 (TYW1) was heterologously expressed in and purified to homogeneity. TYW1 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. TYW1 is the first example of a radical SAM flavoenzyme that is active with NAD(P)H alone.