Iron–Sulfur Cluster
Engineering Provides Insight
into the Evolution of Substrate Specificity among Sulfonucleotide
Reductases
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Abstract
Assimilatory sulfate reduction supplies prototrophic
organisms
with reduced sulfur that is required for the biosynthesis of all sulfur-containing
metabolites, including cysteine and methionine. The reduction of sulfate
requires its activation <i>via</i> an ATP-dependent activation
to form adenosine-5′-phosphosulfate (APS). Depending on the
species, APS can be reduced directly to sulfite by APS reductase (APR)
or undergo a second phosphorylation to yield 3′-phosphoadenosine-5′-phosphosulfate
(PAPS), the substrate for PAPS reductase (PAPR). These essential enzymes
have no human homologue, rendering them attractive targets for the
development of novel antibacterial drugs. APR and PAPR share sequence
and structure homology as well as a common catalytic mechanism, but
the enzymes are distinguished by two features, namely, the amino acid
sequence of the phosphate-binding loop (P-loop) and an iron–sulfur
cofactor in APRs. On the basis of the crystal structures of APR and
PAPR, two P-loop residues are proposed to determine substrate specificity;
however, this hypothesis has not been tested. In contrast to this
prevailing view, we report here that the P-loop motif has a modest
effect on substrate discrimination. Instead, by means of metalloprotein
engineering, spectroscopic, and kinetic analyses, we demonstrate that
the iron–sulfur cluster cofactor enhances APS reduction by
nearly 1000-fold, thereby playing a pivotal role in substrate specificity
and catalysis. These findings offer new insights into the evolution
of this enzyme family and extend the known functions of protein-bound
iron–sulfur clusters