Substitutions in an Active Site Loop of Escherichia coli IscS Result in Specific Defects in Fe-S Cluster and Thionucleoside Biosynthesis in Vivo

Charles T Lauhon,Elizabeth Skovran,Hugo D Urbina,Diana M Downs,Larry E Vickery

doi:10.1074/jbc.m401261200

Abstract

IscS catalyzes the fragmentation of l-cysteine to l-alanine and sulfane sulfur in the form of a cysteine persulfide in the active site of the enzyme. In Escherichia coli IscS, the active site cysteine Cys(328) resides in a flexible loop that potentially influences both the formation and stability of the cysteine persulfide as well as the specificity of sulfur transfer to protein substrates. Alanine-scanning substitution of this 14 amino acid region surrounding Cys(328) identified additional residues important for IscS function in vivo. Two mutations, S326A and L333A, resulted in strains that were severely impaired in Fe-S cluster synthesis in vivo. The mutant strains were deficient in Fe-S cluster-dependent tRNA thionucleosides (s(2)C and ms(2)i(6)A) yet showed wild type levels of Fe-S-independent thionucleosides (s(4)U and mnm(5)s(2)U) that require persulfide formation and transfer. In vitro, the mutant proteins were similar to wild type in both cysteine desulfurase activity and sulfur transfer to IscU. These results indicate that residues in the active site loop can selectively affect Fe-S cluster biosynthesis in vivo without detectably affecting persulfide delivery and suggest that additional assays may be necessary to fully represent the functions of IscS in Fe-S cluster formation.

Highlights

IntroductionE. coli contains iscS and two other paralogs, csdA and sufS (csdB)
IscS is required for the biosynthesis of normal levels of all thionucleosides in E. coli [25] and Salmonella typhimurium [26], two of them are not expected to involve Fe-S cluster enzymes in their synthesis
Generation of IscS Mutants—Alanine-scanning mutagenesis was performed on a 14 amino acid region (Ser323-Ser336) that encompasses the active site loop region of E. coli IscS

Summary

Introduction

E. coli contains iscS and two other paralogs, csdA and sufS (csdB) These enzymes can be placed into two groups, based on sequence of an active site loop that contains an essential cysteine and other criteria [13]. IscS was isolated based on its ability to provide sulfur for a number of different biosynthetic pathways (Fig. 1) [1,2,3,4] It is the Fe-S cluster of dihydroxy acid dehydratase and found to a member of the NifS family of cysteine desulfurases whose have a mechanism similar to NifS [19]. The exact first discovered member, NifS, was characterized as a neces- mechanism of sulfur incorporation in Fe-S clusters remains sary component for activity of the metalloenzyme nitrogenase unclear, several groups have obtained evidence of complexation [5] It was later shown in both Azotobacter vinelandii and and persulfide transfer between IscS and IscU (20 –22).

Methods

Results

Conclusion