Structural and Biochemical Characterization of Staphylococcus aureus Cysteine Desulfurase Complex SufSU

Abstract

In this work, we provide the first in vitro characterization of two essential proteins from Staphylococcus aureus (S. aureus) involved in iron−sulfur (Fe−S) cluster biogenesis: the cysteine desulfurase SufS and the sulfurtransferase SufU. Together, these proteins form the transient SufSU complex and execute the first stage of Fe−S cluster biogenesis in the SUFlike pathway in Gram-positive bacteria. The proteins involved in the SUF-like pathway, such as SufS and SufU, are essential in Gram-positive bacteria since these bacteria tend to lack redundant Fe−S cluster biogenesis pathways. Most previous work characterizing the SUF-like pathway has focused on Bacillus subtilis (B. subtilis). We focus on the SUF-like pathway in S. aureus because of its potential to serve as a therapeutic target to treat S. aureus infections. Herein, we characterize S. aureus SufS (SaSufS) by X-ray crystallography and UV−vis spectroscopy, and we characterize S. aureus SufU (SaSufU) by a zinc binding fluorescence assay and small-angle X-ray scattering. We show that SaSufS is a type II cysteine desulfurase and that SaSufU is a Zn 2+ -containing sulfurtransferase. Additionally, we evaluated the cysteine desulfurase activity of the SaSufSU complex and compared its activity to that of B. subtilis SufSU. Subsequent cross-species activity analysis reveals a surprising result: SaSufS is significantly less stimulated by SufU than BsSufS. Our results set a basis for further characterization of SaSufSU as well as the development of new therapeutic strategies for treating infections caused by S. aureus by inhibiting the SUF-like pathway.

Structural and Biochemical Characterization of Mycobacterium tuberculosis Zinc SufU-SufS Complex

“…This zinc coordination sphere is identical to that previously reported in B. subtilis [24]. His354 is well conserved among SufS in bacteria where the SUF system includes a SufU protein, such as B. subtilis, S. aureus, Myxococcus xantus, and Enterococcus faecalis (Figure S6) [22,[60][61][62].…”

“…unable to bind an Fe-S cluster [61]. Using both Mtb Zn-SufU and apo-SufU proteins, we detected no Fe-S clusters bound to the active site, strongly suggesting that SufU is not an Fe-S protein/scaffold.…”

“…In the E. faecalis system, SufU also stimulates SufS activity, but to a lesser extent (Table 4) [62]. Surprisingly, SufS from S. aureus was recently reported to be poorly stimulated in the presence of its cognate SufU [61]. It is possible that the S. aureus system requires SufBCD in addition to SufU to stimulate SufS, as observed for the E. coli system [16].…”

“…Despite the partial activity with Co 2+ , the low sulfurtransferase activity observed in the presence of other metal ions strongly suggests that Mtb SufU sulfurtransferase activity is zinc-specific. It is worth noting that two other SufU proteins have also been purified with zinc, SufU from B. subtilis and from S. aureus [22,61].…”

“…In contrast, another study claimed that B. subtilis could not act as a standard Fe-S scaffold protein [22,60]. E. faecalis SufU was also reported to bind an Fe-S cluster [61]. Based on these contrasting results, we decided to investigate Mtb SufU as a potential Fe-S binding protein.…”