IscS Functions as a Primary Sulfur-donating Enzyme by Interacting Specifically with MoeB and MoaD in the Biosynthesis of Molybdopterin in Escherichia coli

Wanjiao Zhang,Alexander Urban,Hisaaki Mihara,Silke Leimkühler,Tatsuo Kurihara,Nobuyoshi Esaki

doi:10.1074/jbc.m109.082172

Wanjiao Zhang, Alexander Urban + Show 4 more

Open Access

https://doi.org/10.1074/jbc.m109.082172

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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.

Highlights

Lipoic acid, biotin, iron-sulfur (FeS)2 clusters, and molybdenum cofactors participate in stabilizing reaction intermediates [1, 2], alternating between the reduced dithiol and oxidized disulfide, facilitating electron transfer, or modulating the redox potential of the cofactor [3]
Despite the diverse chemical environments in which the sulfur atoms reside in cofactors and thionucleosides, it has been found that the incorporation of sulfur into them involves a few enzymes with the persulfide (R-S-SH) groups on the cysteine residues of these enzymes; these enzymes act as the key agents of sulfur transfer [8, 9]
The catalytic activity of SufS is significantly lower than that of CsdA and IscS [16], but it is enhanced by SufE through persulfide sulfur transfer; its catalytic activity is further enhanced by the SufBCD complex

Summary

Introduction

Lipoic acid, biotin, iron-sulfur (FeS)2 clusters, and molybdenum cofactors participate in stabilizing reaction intermediates [1, 2], alternating between the reduced dithiol and oxidized disulfide, facilitating electron transfer, or modulating the redox potential of the cofactor [3]. To visualize sulfur flow in the reaction, persulfide forms of the cysteine desulfurases were incubated with MoeB and MoaD under anaerobic conditions and subjected to SDS-PAGE analysis without a reducing agent (Fig. 2).

Results

Conclusion