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

Bradley J. Stevenson, Andy Pranata, Malcolm D. McLeod

    Research output: Contribution to journalArticlepeer-review

    Abstract

    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 β-configured steroid sulfates but like other known class I sulfatases it is inefficient at hydrolyzing α-configured steroid sulfates. We have used directed evolution with liquid chromatography mass spectrometry screening to find variants capable of hydrolyzing a α-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 ~80 times faster than the best PaS variant previously available. This DRN-PaS also shows improved activity for other α-configured steroid sulfates. Simultaneous mutagenesis was essential to obtain DRN-PaS due to complementarity between targeted residues.

    Original languageEnglish
    Article numbergzac007
    JournalProtein Engineering, Design and Selection
    Volume35
    DOIs
    Publication statusPublished - 12 Nov 2022

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