Figure 1.
The reaction catalyzed by Rv3406.
The alpha carbon of an alkyl sulfate is oxidized by Rv3406 in the presence of α-ketoglutarate and spontaneously collapses to an aldehyde and sulfate, liberating CO2 and succinate. The product formation was monitored using a coupled assay using LADH to reduce the aldehyde in a NADH dependent manner.
Figure 2.
Biochemical characterization of Rv3406.
(A) Rv3406 is an αKG and ascorbate dependent sulfatase. Black squares are the complete assay with Rv3406, 2-EHS, αKG, ascorbate and iron. Red triangles are without 2-EHS. Blue circles are without αKG. (B) Rv3406 is an iron dependent enzyme. (C) The rate of Rv3406 accelerates with the addition of ascorbate up to 1 mM. Rv3406 enzyme concentration was between 0.5 and 0.75 µM for all experiments.
Table 1.
Substrates screened for activity in the Rv3406 in vitro coupled assay.
Figure 3.
Analysis of the Rv3406 crystal structure.
(A) Alignment of full length Rv3406 (green) with AtsK (PDB 1OIH, blue) with disordered loops modeled onto the structure. (B) A view of the active site with structures of the substrate and co-substrates modeled from AtsK. (C) Alignment of Rv3406 (green) with E. coli TauD (grey). Loop shown in pink contains the amino acids that hydrogen bond with taurine.
Figure 4.
Protein alignment of alkyl sulfatase enzymes with taurine dioxygenase enzymes.
Enzymes in bold have been biochemically characterized. (A) Alignment of disordered loop 1 where the red boxes are indicating the taurine binding residues in taurine dioxygenases and the analogous amino acids in alkyl sulfatase enzymes. (B) Alignment of disordered loop 2 where the red box is indicating the conserved phenylalanine in taurine dioxygenases and the analogous tyrosine in alkyl sulfate enzymes.
Figure 5.
Rv3406 is essential in Mtb for growth on 2-EHS as the sole sulfur source.
(A) Growth of Mtb strains using either 2-EHS alone or 2-EHS with sodium sulfate. (B) Growth of Mtb strains on n-heptyl sulfate or SDS. Data represents three biological replicates and error bars denote standard deviation. Asterisk indicates a p value of less than 0.005.