The gene thiI encodes a protein (ThiI)
that plays a role in the transfer of sulfur from cysteine
to both thiamin and 4-thiouridine, but the reaction catalyzed
by ThiI remains undetermined. Based upon sequence alignments,
ThiI shares a unique “P-loop” motif with the
PPi synthetase family, four enzymes that catalyze
adenylation and subsequent substitution of carbonyl oxygens.
To test whether or not this motif is critical for ThiI
function, the Asp in the motif was converted to Ala (D189A),
and a screen for in vivo 4-thiouridine production revealed
the altered enzyme to be inactive. Further scrutiny of
sequence data and the crystal structures of two members
of the PPi synthetase family implicated Lys321
in the proposed adenylation function of ThiI, and the critical
nature of Lys321 has been demonstrated by site-directed
mutagenesis and genetic screening. Our results, then, indicate
that ThiI catalyzes the adenylation of a substrate at the
expense of ATP, a narrowing of possible reactions that
provides a strong new basis for deducing the early steps
in the transfer of sulfur from cysteine to both thiamin
and 4-thiouridine.