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J Biol Chem, Vol. 275, Issue 15, 10727-10730, April 14, 2000
From the School of Pharmacy, University of Wisconsin, Madison,
Wisconsin 53706
IscS from Escherichia coli is a
cysteine desulfurase that has been shown to be involved in Fe-S cluster
formation. The enzyme converts L-cysteine to
L-alanine and sulfane sulfur (S0) in the form
of a cysteine persulfide in its active site. Recently, we reported that
IscS can donate sulfur for the in vitro biosynthesis of
4-thiouridine (s4U), a modified nucleotide in tRNA. In
addition to IscS, s4U synthesis in E. coli also
requires the thiamin biosynthetic enzyme ThiI, Mg-ATP, and
L-cysteine as the sulfur donor. We now report evidence that
the sulfane sulfur generated by IscS is transferred sequentially to
ThiI and then to tRNA during the in vitro synthesis of
s4U. Treatment of ThiI with
5-((2-iodoacetamido)ethyl)-1-aminonapthalene sulfonic acid (I-AEDANS)
results in irreversible inhibition, suggesting the presence of a
reactive cysteine that is required for binding and/or catalysis. Both
ATP and tRNA can protect ThiI from I-AEDANS inhibition. Finally, using
gel shift and protease protection assays, we show that ThiI binds to
unmodified E. coli tRNAPhe. Together, these
results suggest that ThiI is a recipient of S0 from IscS
and catalyzes the ultimate sulfur transfer step in the biosynthesis of
s4U.
ACCELERATED PUBLICATION
Evidence for the Transfer of Sulfane Sulfur from IscS to ThiI
during the in Vitro Biosynthesis of 4-Thiouridine in
Escherichia coli tRNA*
*
This work was supported by National Institutes of Health
Grant GM57002 (to C. T. L.) and by a grant from the University of Wisconsin Graduate School.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
To whom correspondence should be addressed: School of Pharmacy,
University of Wisconsin, 425 N. Charter St., Madison, WI 53706. Tel.:
608-262-3083; Fax: 608-262-3397; E-mail:
clauhon@facstaff.wisc.edu.
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