Lauhon Charles T, Skovran Elizabeth, Urbina Hugo D, Downs Diana M, Vickery Larry E
School of Pharmacy, University of Wisconsin, Madison, WI 53705, USA.
J Biol Chem. 2004 May 7;279(19):19551-8. doi: 10.1074/jbc.M401261200. Epub 2004 Feb 21.
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.
IscS在酶的活性位点催化L-半胱氨酸断裂生成L-丙氨酸和以半胱氨酸过硫化物形式存在的硫烷硫。在大肠杆菌IscS中,活性位点半胱氨酸Cys(328)位于一个柔性环中,该柔性环可能会影响半胱氨酸过硫化物的形成与稳定性,以及硫向蛋白质底物转移的特异性。对围绕Cys(328)的这14个氨基酸区域进行丙氨酸扫描取代,确定了体内IscS功能的其他重要残基。两个突变,S326A和L333A,导致菌株在体内铁硫簇合成中严重受损。突变菌株缺乏铁硫簇依赖性的tRNA硫代核苷(s(2)C和ms(2)i(6)A),但显示出野生型水平的不依赖铁硫簇的硫代核苷(s(4)U和mnm(5)s(2)U),后者需要过硫化物的形成和转移。在体外,突变蛋白在半胱氨酸脱硫酶活性和向IscU的硫转移方面与野生型相似。这些结果表明,活性位点环中的残基可以在体内选择性地影响铁硫簇生物合成,而不会显著影响过硫化物的传递,并表明可能需要额外的测定来全面表征IscS在铁硫簇形成中的功能。