Direct observation of intermediates in the SufS cysteine desulfurase reaction reveals functional roles of conserved active-site residues

Abstract

Iron-sulfur (Fe-S) clusters are necessary for the proper functioning of numerous metalloproteins. Fe-S cluster (Isc) and sulfur utilization factor (Suf) pathways are the key biosynthetic routes responsible for generating these Fe-S cluster prosthetic groups in Escherichia coli. Although Isc dominates under normal conditions, Suf takes over during periods of iron depletion and oxidative stress. Sulfur acquisition via these systems relies on the ability to remove sulfur from free cysteine using a cysteine desulfurase mechanism. In the Suf pathway, the dimeric SufS protein uses the cofactor pyridoxal 5-phosphate (PLP) to abstract sulfur from free cysteine, resulting in the production of alanine and persulfide. Despite much progress, the stepwise mechanism by which this PLP-dependent enzyme operates remains unclear. Here, using rapid-mixing kinetics in conjunction with X-ray crystallography, we analyzed the pre-steadystate kinetics of this process while assigning early intermediates of the mechanism. We employed H123A and C364A SufS variants to trap Cys-aldimine and Cys-ketimine intermediates of the cysteine desulfurase reaction, enabling direct observations of these intermediates and associated conformational changes of the SufS active site. Of note, we propose that Cys-364 is essential for positioning the Cys-aldimine for C␣ deprotonation, His-123 acts to protonate the Ala-enamine intermediate, and Arg-56 facilitates catalysis by hydrogen bonding with the sulfhydryl of Cys-aldimine. Our results, along with previous SufS structural findings, suggest a detailed model of the SufS-catalyzed reaction from Cys binding to C-S bond cleavage and indicate that Arg-56, His-123, and Cys-364 are critical SufS residues in this C-S bond cleavage pathway.

Snapshots of PLP‐substrate and PLP‐product external aldimines as intermediates in two types of cysteine desulfurase enzymes

“…These interactions, especially found between the conserved His and the thiol group of PLP-L-cysteine, are beneficial for a nucleophilic attack to the thiol group of PLP-L-cysteine ketimine, which is converted from the corresponding aldimine, by the catalytic cysteine on the loop. The finding of interaction between the His and the thiol group in the present study is consistent with the results of the observation of the interaction in PLP-L-cysteine ketimine of the SufS variant in a recent study [20]. It should be noted that the PLP-L-cysteine and PLP-L-alanine aldimines in both NifS (type I) and SufS (type II) are in almost the same conformations with key interactions between the conserved histidine residues and the thiol groups.…”

“…The observed key interactions (e.g., interaction of conserved His-thiol group of PLP-L-cysteine) allowed to confirm the revised reaction mechanism (summarized in Fig. 9, including His-thiol interaction and difference of the loop motions in type I and II enzymes), which has also recently been demonstrated with X-ray crystallography on SufS variants and stopped-flow analysis by Outten et al [20]. Moreover, the intermediates were found in the identical conformations in both type I and type II, which indicated that the cysteine desulfurase enzymes commonly use the same intermediates based on PLP.…”

“…(C) Revised catalytic mechanisms of type I and II cysteine desulfurase enzymes with consideration of His‐thiol interaction and loop structural changes. This revised mechanism is proposed by referring to a revised catalytic mechanism based on recent study of E. coli SufS (type II), proposed by Outten and coworkers . In other words, the revised mechanism of E. coli SufS is expanded to type I enzymes with some modifications based on the present study.…”

“…Indeed, this loop was shown to be disordered in previous studies on the crystal structures of type I cysteine desulfurase enzymes . On the other hand, the corresponding loop of type II cysteine desulfurase SufS is formed as an α‐helix in the resting state, which has been visualized by X‐ray crystal structure analysis .…”

Exploring the selenium-over-sulfur substrate specificity and kinetics of a bacterial selenocysteine lyase

“…2C). Recently, it has been found that H123 of E. coli SufS is required for CDS activity and that it may function as an acid-base catalyst in this mechanism (57). Moreover, the authors believed that H123 could deprotonate the catalytic thiol of C364 and share a π-π stacking interaction with PLP (57).…”