IscS functions as a primary sulfur-donating enzyme by interacting specifically with MoeB and MoaD in the biosynthesis of molybdopterin in Escherichia coli

Abstract

The persulfide sulfur formed on an active site cysteine residue of pyridoxal 5'-phosphate-dependent cysteine desulfurases is subsequently incorporated into the biosynthetic pathways of a variety of sulfur-containing cofactors and thionucleosides. In molybdenum cofactor biosynthesis, MoeB activates the C terminus of the MoaD subunit of molybdopterin (MPT) synthase to form MoaD-adenylate, which is subsequently converted to a thiocarboxylate for the generation of the dithiolene group of MPT. It has been shown that three cysteine desulfurases (CsdA, SufS, and IscS) of Escherichia coli can transfer sulfur from l-cysteine to the thiocarboxylate of MoaD in vitro. Here, we demonstrate by surface plasmon resonance analyses that IscS, but not CsdA or SufS, interacts with MoeB and MoaD. MoeB and MoaD can stimulate the IscS activity up to 1.6-fold. Analysis of the sulfuration level of MoaD isolated from strains defective in cysteine desulfurases shows a largely decreased sulfuration level of the protein in an iscS deletion strain but not in a csdA/sufS deletion strain. We also show that another iscS deletion strain of E. coli accumulates compound Z, a direct oxidation product of the immediate precursor of MPT, to the same extent as an MPT synthase-deficient strain. In contrast, analysis of the content of compound Z in DeltacsdA and DeltasufS strains revealed no such accumulation. These findings indicate that IscS is the primary physiological sulfur-donating enzyme for the generation of the thiocarboxylate of MPT synthase in MPT biosynthesis.

FIGURE 1.

Proposed pathway for the biosynthesis of molybdenum cofactor in E. coli. The structures of precursor Z, MPT, molybdenum cofactor, and the oxidation product of precursor Z, i.e. compound Z, are shown. The incorporation of sulfur atoms into precursor Z is catalyzed by MPT synthase (MoaE/MoaD). Three cysteine desulfurases (CsdA, SufS, and IscS) transfer the sulfur atom from l-cysteine to the C-terminal thiocarboxylate of MoaD in vitro (36, 41).

FIGURE 2.

Analysis of the sulfur transfer from cysteine desulfurases to MoaD by ³⁵S-labeled l-cysteine. Lane 1, CsdA; lane 2, CsdA and MoeB; lane 3, CsdA and MoaD; lane 4, CsdA, MoeB, and MoaD; lane 5, CsdA, MoeB, and MoaD (without ATP); lane 6, SufS; lane 7, SufS and MoeB; lane 8, SufS and MoaD; lane 9, SufS, MoeB, and MoaD; lane 10, SufS, MoeB, and MoaD (without ATP); lane 11, IscS; lane 12, IscS and MoeB; lane 13, IscS and MoaD; lane 14, IscS, MoeB, and MoaD; lane 15, IscS, MoeB, and MoaD (without ATP). The gel was stained with Coomassie Brilliant Blue (A), and the ³⁵S radioactivity was visualized on a phosphorimaging plate (B).

FIGURE 3.

Activation of cysteine desulfurase activity by MoaD or MoeB. Formations of sulfide from l-cysteine were measured after 30 min for CsdA (A) and IscS (C) and 90 min for SufS (B).

FIGURE 4.

Surface plasmon resonance analysis to examine the interaction between MoaD (A) or MoeB (B) and three cysteine desulfurases of E. coli.

FIGURE 5.

Detection of the sulfuration level of MoaD after expression in different E. coli mutant strains. The sulfuration level of MoaD was determined by the amount of MPT formed after incubation of purified MoaD, MoaE, and precursor Z in excess. The amount of MPT formed during the reaction was quantified as Form A using the peak height.

FIGURE 6.

Analysis of the acid extracts of E. coli cells. The acid extracts of the wild-type, ΔcsdA, ΔsufS, ΔiscS, and ΔmoaD strains were analyzed using HPLC with a Capcell Pak C₁₈ SG120 column after air oxidation. The volumes of the analyzed samples were 12.5 μl for the ΔmoaD strain and 25 μl for the other strains.