Crystal structure of YnjE from Escherichia coli, a sulfurtransferase with three rhodanese domains

Abstract

Rhodaneses/sulfurtransferases are ubiquitous enzymes that catalyze the transfer of sulfane sulfur from a donor molecule to a thiophilic acceptor via an active site cysteine that is modified to a persulfide during the reaction. Here, we present the first crystal structure of a triple-domain rhodanese-like protein, namely YnjE from Escherichia coli, in two states where its active site cysteine is either unmodified or present as a persulfide. Compared to well-characterized tandem domain rhodaneses, which are composed of one inactive and one active domain, YnjE contains an extra N-terminal inactive rhodanese-like domain. Phylogenetic analysis reveals that YnjE triple-domain homologs can be found in a variety of other c-proteobacteria, in addition, some single-, tandem-, four and even six-domain variants exist. All YnjE rhodaneses are characterized by a highly conserved active site loop (CGTGWR) and evolved independently from other rhodaneses, thus forming their own subfamily. On the basis of structural comparisons with other rhodaneses and kinetic studies, YnjE, which is more similar to thiosulfate:cyanide sulfurtransferases than to 3-mercaptopyruvate:cyanide sulfurtransferases, has a different substrate specificity that depends not only on the composition of the active site loop with the catalytic cysteine at the first position but also on the surrounding residues. In vitro YnjE can be efficiently persulfurated by the cysteine desulfurase IscS. The catalytic site is located within an elongated cleft, formed by the central and C-terminal domain and is lined by bulky hydrophobic residues with the catalytic active cysteine largely shielded from the solvent.

Plant Pathogenic Bacteria Utilize Biofilm Growth-associated Repressor (BigR), a Novel Winged-helix Redox Switch, to Control Hydrogen Sulfide Detoxification under Hypoxia

“…Thus, we searched for proteins having the C-(X) 4 -R consensus and found that rhodaneses (sulfurtransferases) have highly conserved active-site loops with a CRXGX(R/T) motif (47). Surprisingly, the superposition of the Blh DUF442 with the catalytic domain of E. coli rhodanese YnjE (21) shows that the two domains have a similar fold and a remarkable conservation of the active-site residues, including the catalytic cysteine (supplemental Fig. S4), suggesting that DUF442 could display a rhodanese-like activity.…”

“…Nevertheless, although alternating cycles of model building using COOT (12) and refinement with REFMAC5 (13) allowed the modeling of 96 out of the 102 residues expected in the asymmetric unit (a single monomer), R work /R free values remained at 0.230/0.263, and the addition of water molecules caused the R free to rise. Refinement was done against both native and derivative data processed in the space group P3 1 21, and the same problem was encountered. At this point, lower symmetry space groups were applied, and structure refinement could only be accomplished in P1.…”

Solution NMR Structure and Functional Analysis of the Integral Membrane Protein YgaP from Escherichia coli

“…The enzymatic reaction takes place in two steps. In the first step, the thiol group of the cysteine reacts with the thiosulfate anion (S 2 O 3 Ϫ ) to form an enzyme-persulfide intermediate, which reacts in a second step with the cyanide ion to produce thiocyanate (SCN Ϫ ) (7)(8)(9). Although the crystal structure of the sulfur-free and the persulfide form of one member of the rhodanese family (GlpE) as well as the solution state NMR structure of another member (PspE) have been determined (3,4,10), the molecular mechanism of the sulfurtransferase activity remains unclear (11), but the dynamics appear to be involved (3).…”

The Identification of a Novel Protein Involved in Molybdenum Cofactor Biosynthesis in Escherichia coli

“…In each case, only the C-terminal domain contains the catalytic cysteine residue. The crystal structure of YnjE has been solved recently (19). YnjE can efficiently be persulfurated by IscS while having only a residual activity with thiosulfate as substrate (19).…”

“…The crystal structure of YnjE has been solved recently (19). YnjE can efficiently be persulfurated by IscS while having only a residual activity with thiosulfate as substrate (19). The role of YnjE so far remained enigmatic for E. coli.…”