Improving the arylsulfatase from Pseudomonas aeruginosa for anti-doping applications
Creators: name: Uduwela, Dimanthi Roshika
Publisher: name: The Australian National University
Year: 2017
Subjects: Pseudomonas aeruginosa arylsulfatase, semi-rational design, steroid sulfates, anti-doping, LC-MS

description: The misuse of anabolic androgenic steroids in sports remains a significant concern. They are exploited to enhance physical performance, and as a result, they are listed as prohibited substances by the world anti-doping agency (WADA). Detection of steroids is important to maintain fair play in sports and to ensure that athletes are not jeopardising their long-term health. The human phase II steroid metabolites are mainly excreted in urine as glucuronide or sulfate conjugates. The routine screening methods require deconjugation of the phase II conjugates to produce the free steroids. The hydrolysis of steroid glucuronides is efficiently achieved by using E. coli β-glucuronidase enzyme. However, there is no robust method for hydrolysing steroid sulfates. Acid catalysed solvolysis is a general method of hydrolysis of steroid conjugates. However, this method is often unsuitable for routine anti-doping screening since it can degrade analytes of interest and generate a more complicated analytical matrix. Steroid sulfates are emerging as highly useful markers to detect AAS abuse, importantly as long-term markers that have the potential to improve the sensitivity and the retrospectivity of steroid screening. However, their routine detection would benefit from a robust steroid sulfatase for deconjugation, in a manner analogous to the β-glucuronidase enzyme. The arylsulfatase from Pseudomonas aeruginosa (PaS) is an enzyme capable of hydrolysing steroid sulfates and represents a good starting point for engineering a steroid sulfatase. However, this enzyme requires improvement in hydrolytic activity and substrate scope in order to be useful in an anti-doping context. This study aimed to improve the catalytic rate of PaS for the hydrolysis of steroid sulfates such as testosterone sulfate (TS) and to improve the substrate scope while maintaining the stability of the enzyme. These improvements were sought by applying semi-rational design to mutate amino acid residues neighbouring the enzyme active site by taking use of the crystal structure of PaS (1HDH) and the binding modes suggested by computational ligand protein docking. Mutagenesis was implemented on single residues and multiple residue sites and finally shuffling all the beneficial mutations. Screening was performed to test the steroid sulfate hydrolysis activity of these mutant libraries by employing LC-MS. These libraries revealed the steroid sulfate binding pocket of PaS and screening of &gt;10000 variants resulted in three mutants that showed an improvement in the catalytic efficiency (Vmax/ KM) of more than 150 times that of wild type PaS for TS hydrolysis. In addition, the substrate scope of steroid sulfates for PaS enzyme was increased and a modest improvement in thermostability was observed. The mechanistic involvement of E74 as a catalytic residue was proposed with the observation of mutants created on this residue. To apply the enzyme on real world samples, an untargeted screen was performed on pre- and post-administration testosterone propionate (TP) horse urine samples which identified three metabolites that could be potential markers for TP administration. This also revealed that PaS is compatible with urine matrices under the reaction conditions analogous to β-glucuronidase treatment in the anti-doping screens, making it a valuable steroid sulfatase for anti-doping applications.