Engineering Pseudomonas aeruginosa arylsulfatase for hydrolysis of α-configured steroid sulfates

Steroid sulfate esters are important metabolites for anti-doping efforts in sports, pathology and research. Analysis of these metabolites is facilitated by hydrolysis using either acid or enzymatic catalysis. Although enzymatic hydrolysis is preferred for operating at neutral pH, no known enzyme is capable of hydrolyzing all steroid sulfate metabolites. Pseudomonas aeruginosa arylsulfatase (PaS) is ideal for the hydrolysis of beta-configured steroid sulfates but like other known class I sulfatases it is inefficient at hydrolyzing alpha-configured steroid sulfates. We have used directed evolution with liquid chromatography mass spectrometry screening to find variants capable of hydrolyzing a alpha-configured steroid sulfate: etiocholanolone sulfate (ECS). After targeting two regions of PaS, four residues were identified and optimized to yield a final variant with a total of seven mutations (DRN-PaS) capable of hydrolyzing ECS similar to 80 times faster than the best PaS variant previously available. This DRN-PaS also shows improved activity for other alpha-configured steroid sulfates. Simultaneous mutagenesis was essential to obtain DRN-PaS due to complementarity between targeted residues. [GRAPHICS] .

Automated summary

Steroid sulfate esters are important metabolites for various fields. Researchers used directed evolution to discover a variant, DRN-PaS, capable of efficiently hydrolyzing alpha-configured steroid sulfates, with improved activity compared to previous variants.