Structure of an archaeal TYW1, the enzyme catalyzing the second step of wye-base biosynthesis

Abstract

Wye bases are tricyclic bases that are found in archaeal and eukaryotic tRNAs. The most modified wye base, wybutosine, which appears at position 37 (the 3'-adjacent position to the anticodon), is known to be important for translational reading-frame maintenance. Saccharomyces cerevisiae TYW1 catalyzes the tri-ring-formation step in wye-base biosynthesis, with the substrate tRNA bearing N(1)-methylated G37. Here, the crystal structure of the archaeal TYW1 homologue from Pyrococcus horikoshii is reported at 2.2 A resolution. The amino-acid sequence of P. horikoshii TYW1 suggested that it is a radical-AdoMet enzyme and the tertiary structure of P. horikoshii TYW1 indeed shares the modified TIM-barrel structure found in other radical-AdoMet enzymes. Radical-AdoMet enzymes generally contain one or two iron-sulfur (FeS) clusters. The tertiary structure of P. horikoshii TYW1 revealed two FeS cluster sites, each containing three cysteine residues. One FeS cluster site was expected from the amino-acid sequence and the other involves cysteine residues that are dispersed throughout the sequence. The existence of two FeS clusters was confirmed from the anomalous Fourier electron-density map. By superposing the P. horikoshii TYW1 tertiary structure on those of other radical-AdoMet enzymes, the AdoMet molecule, which is necessary for the reactions of radical-AdoMet enzymes, was modelled in P. horikoshii TYW1. Surface plots of conservation rates and electrostatic potentials revealed the highly conserved and positively charged active-site hollow. On the basis of the surface properties, a docking model of P. horikoshii TYW1, the tRNA, the FeS clusters and the AdoMet molecule was constructed, with the nucleoside at position 37 of tRNA flipped out from the canonical tRNA structure.

Wybutosine biosynthesis: Structural and mechanistic overview

“…This suggests that in addition to the tRNA substrate and SAM co-substrate, a second co-substrate, which is a source of the inserted 2-carbon unit, is also involved in the reaction. 6 TYW1, the enzyme that catalyzes the transformation of m 1 G 37 into ImG-14 has previously been classified as a Radical-SAM enzyme due to the presence of a conserved CysxxxCysxxCys motif in its aminoacid sequence 6 In 2007, 2 apo structures of TYW1 from Methanococcus jannaschii and Pyrococcus horikoshii have been reported 30,31 ( Fig. 8A & B).…”

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

“…The native, mostly apo forms of TYW1 enzyme from Methanococcus jannaschii and Pyrococcus horikoshii, have been crystallized and their structures reported (13,14). Despite the fact that no electron density for the cluster atoms could be observed in the crystal structure, it was suggested from data collection and phasing statistics that a spatial configuration in which the sulfhydryl groups of the CX 12 CX 12 C motif is optimal for chelating another [4Fe-4S] cluster (herein called cluster II).…”

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

“…In TYW1 these residues are located in the presumed active site across from the Cys residues that constitute the radical SAM motif. The structures do not show clear and unambiguous density for either of the metalloclusters [25,26]. In vivo complementation studies, however, suggest that both sets of Cys residues are essential for activity [25].…”

“…While the essential role of TYW1 in forming yW was established early and several structures of the protein had been available, in vitro reconstitution of its activity had eluded the field until recently [4,15,25,26]. The imidazoline ring of yW is built on the m 1 G backbone of tRNA, suggesting that a 2-carbon donor molecule was involved in the reaction.…”