Structural and mechanistic insights into the PAPS-independent sulfotransfer catalyzed by bacterial aryl sulfotransferase and the role of the DsbL/Dsbl system in its folding.

Bacterial aryl sulfotransferases catalyze sulfotransfer from a phenolic sulfate to a phenol. These enzymes are frequently found in pathogens and upregulated during infection. Their mechanistic understanding is very limited, and their natural substrates are unknown. Here, the crystal structures of Escherichia coli CFT073 trapped in its presulfurylation state with model donor substrates bound in the active site are reported, which reveal the molecular interactions governing substrate recognition. Furthermore, spectroscopic titrations with donor substrates and sulfurylation kinetics of Aduanyu1942 illustrate that this enzyme binds substrates in a 1:1 stoichiometry and that the active sites of the Aduanyu1942 homooligomer act independently. Mass spectrometry and crystallographic experiments of Aduanyu1942 incubated with human urine demonstrate that urine contains a sulfuryl donor substrate. In addition, we examined the capability of the two paralogous dithiol oxidases present in uropathogenic E. coli CFT073, DsbA, and the enzyme DsbL, to introduce the single, conserved disulfide bond into We show that DsbA and DsbL introduce the disulfide bond into unfolded Aduanyu1942 at similar rates. Hence, a chaperone effect of DsbL, not present in DsbA, appears to be responsible for the dependence of efficient Aduanyu1942 folding on DsbL in vivo. The conservation of paralogous dithiol oxidases with different substrate specificities in certain bacterial strains may therefore be a consequence of the complex folding pathways of their substrate proteins.

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