Structural Evidence for a [4Fe‐5S] Intermediate in the Non‐Redox Desulfuration of Thiouracil

Abstract

We recently discovered a [Fe-S]-containing protein with in vivo thiouracil desulfidase activity called TudS. We report here the crystal structure of TudS, refined at 1.5 Å resolution, which harbors a [4Fe-4S] cluster, bound by three cysteines only. Incubation of TudS crystals with 4thiouracil trapped the cluster with a hydrosulfide ligand bound to the fourth non-protein-bonded iron, as established by the sulfur anomalous signal. This indicates that a [4Fe-5S] state of the cluster is a catalytic intermediate in the desulfuration reaction. Structural data and site-directed mutagenesis indicate that a water molecule is located next to the hydrosulfide ligand and to two catalytically important residues, Ser101 and Glu45. This information together with modeling studies allow us to propose a mechanism for the unprecedented nonredox enzymatic desulfuration of thiouracil, in which a [4Fe-4S] cluster binds and activates the sulfur atom of the substrate.

Prediction of the Iron–Sulfur Binding Sites in Proteins Using the Highly Accurate Three-Dimensional Models Calculated by AlphaFold and RoseTTAFold

“…2.6. The Fe-S-Binding Site Is Pre-Formed in the 3D Model of Cysteine Desulfidase CyuA, but the Cluster Coordination Is Ambiguous Desulfidases catalyze the desulfuration of a substrate using a [4Fe-4S] cluster [44,45], which is the inverse reaction of tRNA sulfuration as discussed previously. There are very few proteins from this family that have been characterized and only one structure of one of these enzymes in a holo-form was reported from our group [45].…”

TtuA and TudS, Two [4Fe‐4S]‐Dependent Enzymes Catalyzing Nonredox Sulfuration or Desulfuration Reactions

“…Using an uracil auxotroph selection system in E. coli, metagenomic libraries from soil samples were screened for genes allowing cellular growth on a minimal medium containing 2-thiouracil 11 . The gene product was named TudS -for ThioUracil DeSulfidase -after the confirmation of its in vitro catalytic activity 13 .…”

Determination of the Absolute Molar Mass of [Fe-S]-Containing Proteins Using Size Exclusion Chromatography-Multi-Angle Light Scattering (SEC-MALS)

“…Purification of human CISD2, Escherichia coli MnmA, Aeromonas TudS and human mitoNEET has been described previously [ 26 , 32 , 35 , 37 ], whereas the overexpression and purification of LarE and CyuA from Methanococcus maripaludis will be reported elsewhere (unpublished results). MitoNEET and CISD2 were purified aerobically as holo-proteins, whereas chemical cluster reconstitution was performed anaerobically with the as-purified proteins (mixture of apo/holo forms) of MnmA, TudS, LarE and CyuA, as described previously [ 32 ].…”

“…On the other hand, sulfuration enzymes and desulfidases are [4Fe-4S]-dependent enzymes. tRNA sulfuration enzymes are involved in the biosynthesis of sulfur-containing nucleosides, which are essential for the efficiency and accuracy of genetic translation [ 30 , 31 , 32 , 33 ], whereas LarE catalyzes sulfur insertion in the cofactor of lactate racemase [ 34 ], TudS is a recently discovered thiouracil desulfidase [ 35 ] and CyuA is a cysteine desulfidase [ 36 ]. In the case of these sulfurases and desulfidases, the [4Fe-4S] cluster is bound by three ligands only, and the fourth non-protein bonded unique iron is presumed to bind and activate the sulfur donor for the sulfuration reaction [ 30 , 31 ] or the sulfur of the substrate for the desulfuration reaction [ 35 ].…”