A subclass of archaeal U8-tRNA sulfurases requires a [4Fe–4S] cluster for catalysis

Abstract

Sulfuration of uridine 8, in bacterial and archaeal tRNAs, is catalyzed by enzymes formerly known as ThiI, but renamed here TtuI. Two different classes of TtuI proteins, which possess a PP-loop-containing pyrophosphatase domain that includes a conserved cysteine important for catalysis, have been identified. The first class, as exemplified by the prototypic Escherichia coli enzyme, possesses an additional C-terminal rhodanese domain harboring a second cysteine, which serves to form a catalytic persulfide. Among the second class of TtuI proteins that do not possess the rhodanese domain, some archaeal proteins display a conserved CXXC + C motif. We report here spectroscopic and enzymatic studies showing that TtuI from Methanococcus maripaludis and Pyrococcus furiosus can assemble a [4Fe–4S] cluster that is essential for tRNA sulfuration activity. Moreover, structural modeling studies, together with previously reported mutagenesis experiments of M. maripaludis TtuI, indicate that the [4Fe–4S] cluster is coordinated by the three cysteines of the CXXC + C motif. Altogether, our results raise a novel mechanism for U8-tRNA sulfuration, in which the cluster is proposed to catalyze the transfer of sulfur atoms to the activated tRNA substrate.

The thiolation of uridine 34 in tRNA, which controls protein translation, depends on a [4Fe-4S] cluster in the archaeum Methanococcus maripaludis

“…The first mechanism involves intermediate reactive persulfides carried by protein cysteines, as exemplified by the biosynthesis of s 4 U8 in tRNAs in E. coli , in which sulfur is released from free cysteine by cysteine desulfurase IscS, transferred to the rhodanese-like module of the U8-tRNA thiolase as a persulfide, which finally gives its sulfur atom, directly or indirectly, to the adenylated nucleoside 64 , 65 . In contrast, the biosynthesis of s 4 U8-tRNAs in several archaea 45 , 66 , s 2 U34-tRNA catalyzed by bacterial MnmAs 36 , 37 , s 2 C32-tRNA catalyzed by TtcAs 38 and m 5 s 2 U54-tRNA catalyzed by TtuAs 39 – 42 occurs via a mechanism that involves a [4Fe-4S] cluster as a cofactor. The [4Fe-4S] cluster is coordinated by three conserved amino acids only, which leaves a free coordination site on one iron atom to bind and activate the sulfur atom, coming from inorganic sulfide 39 or a sulfur donor 41 , to promote the sulfur transfer to the adenylated substrate (Fig.…”

Molecular mechanism of tRNA binding by the Escherichia coli N7 guanosine methyltransferase TrmB

“…Thereby, we report that the model bacterium E. coli is sensitive to hydrogen peroxide stress in the absence of TrmB. In order to gain a better understanding as to how this tRNA modification enzyme binds tRNA, we prepared partially modified tRNA Phe containing the 4-thiouridine 8 (s 4 U8) modification using purified ThiI (also known as TtuI ( 28 , 29 )) and IscS enzymes. The reactivity of the s 4 U8 modification was then used to fluorescently label the tRNA for presteady-state kinetic analysis using a stopped-flow instrument.…”

TudS desulfidases recycle 4-thiouridine-5’-monophosphate at a catalytic [4Fe-4S] cluster

“…5B ). For this purpose, we isolated bulk tRNA from the wild type, the ΔtudS_KT knockout, and from a ΔthiI knockout, in which a gene encoding the tRNA sulfur transferase involved in 4-thiouridine formation 9 , 36 is deleted, and the 4-thiouridine content of each strain was determined by mass spectrometry. As expected, the content of 4-thiouridine in bulk tRNA was drastically reduced in the ΔthiI knockout strain (negative control).…”