4-Demethylwyosine Synthase from Pyrococcus abyssi Is a Radical-S-adenosyl-l-methionine Enzyme with an Additional [4Fe-4S]+2 Cluster That Interacts with the Pyruvate Co-substrate

Abstract

Background: 4-Demethylwyosine synthase (TYW1) is a tRNA-modifying metalloenzyme involved in the biosynthesis of wyosine. Results: TYW1 enzyme belongs to the Radical-SAM superfamily with two Fe-S clusters involved in catalysis. Conclusion:The canonical Radical-SAM cluster binds and activates SAM co-factor, whereas the additional [4Fe-4S] cluster is shown to interact with the pyruvate co-substrate. Significance: This study helps to understand how radical-SAM enzymes with two Fe-S centers can synergistically achieve challenging radical insertion reactions.

Wybutosine biosynthesis: Structural and mechanistic overview

“…32 More recently, important insights into the mechanisms of TYW1 have been obtained. 33 First, reconstituted TYW1 was shown to contain 2 oxygen-sensitive [4Fe¡4S] 2C/C clusters, each ligated by only 3 cysteine residues that are absolutely required for activity. 33 Second, the N-terminal [4Fe-4S] cluster (cluster II), bound to the polypeptide by a CysX 12 CysX 12 Cys motif, contains a non-cysteinyl iron that is capable to bind and activate the pyruvate co-substrate.…”

“…33 First, reconstituted TYW1 was shown to contain 2 oxygen-sensitive [4Fe¡4S] 2C/C clusters, each ligated by only 3 cysteine residues that are absolutely required for activity. 33 Second, the N-terminal [4Fe-4S] cluster (cluster II), bound to the polypeptide by a CysX 12 CysX 12 Cys motif, contains a non-cysteinyl iron that is capable to bind and activate the pyruvate co-substrate. 33 Third, using a tRNA Phe transcript from S. cerevisiae, which was labeled with ( 13 C 2 H 3 )-methyl-SAM on m 1 G in a Trm5-dependent reaction, m 1 G was shown to be the true substrate of the initial Ado reaction that leads to the formation of imG-14 (T. Molle et al, to be published).…”

“…33 Second, the N-terminal [4Fe-4S] cluster (cluster II), bound to the polypeptide by a CysX 12 CysX 12 Cys motif, contains a non-cysteinyl iron that is capable to bind and activate the pyruvate co-substrate. 33 Third, using a tRNA Phe transcript from S. cerevisiae, which was labeled with ( 13 C 2 H 3 )-methyl-SAM on m 1 G in a Trm5-dependent reaction, m 1 G was shown to be the true substrate of the initial Ado reaction that leads to the formation of imG-14 (T. Molle et al, to be published). These findings place substantial constraints on the mechanism by which the imidazoline ring is formed, as depicted by a working model in Scheme 4.…”

Biosynthesis of wyosine derivatives in tRNAPhe of Archaea: role of a remarkable bifunctional tRNAPhe:m1G/imG2 methyltransferase

“…However, at variance with the yeast Tyw1, archaeal Taw1 does not have the flavin-binding domain but contains a second Fe-S cluster instead (Perche-Letuvée et al 2012). The corresponding recombinant Taw1 enzymes of Pyrococcus horikoschii (PH1705), Methanocaldococcus jannaschii (MJ0257), and Pyrococcus abyssi (PAB2039) have been purified; and in the two last cases, their enzymatic activities have been successfully demonstrated in vitro using pyruvate as the key substrate, AdoMet as the cofactor, and dithionite as the source of reducing power (Goto-Ito et al 2007;Young and Bandarian 2011;Perche-Letuvée et al 2012).…”

Radical mediated ring formation in the biosynthesis of the hypermodified tRNA base wybutosine

“…TYW1, however, utilizes SAM as an essential cofactor to initiate a complex radical mediated transformation. This review will focus on recent insights on the reaction catalyzed by this TYW1 where recent advances in in vitro reconstitution of its activity have made mechanistic studies possible [18,19]. …”