Improving the arylsulfatase from Pseudomonas aeruginosa for anti-doping applications

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 >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

Enhancing the Steroid Sulfatase Activity of the Arylsulfatase from Pseudomonas aeruginosa

Steroidal sulfate esters play a central role in many physiological processes. They serve as the reservoir for endogenous sex hormones and form a significant fraction of the steroid metabolite pool. The analysis of steroid sulfates is thus essential in fields such as medical science and sports drug testing. Although the direct detection of steroid sulfates can be readily achieved using liquid chromatography-mass spectrometry, many analytical approaches, including gas chromatography-mass spectrometry, are hampered due to the lack of suitable enzymatic or chemical methods for sulfate ester hydrolysis prior to analysis. Enhanced methods of steroid sulfate hydrolysis would expand analytical possibilities for the study of these widely occurring metabolites. The arylsulfatase from Pseudomonas aeruginosa (PaS) is a purified enzyme capable of hydrolysing steroid sulfates. However, this enzyme requires improvement to hydrolytic activity and substrate scope in order to be useful in analytical applications. These improvements were sought by applying semi-rational design to mutate amino acid residues neighbouring the enzyme active site. Mutagenesis was implemented on both single and multiple residue sites. Screening by UPLC-MS was performed to test the steroid sulfate hydrolysis activity of these mutant libraries against testosterone sulfate. This approach revealed the steroid sulfate binding pocket and resulted in three mutants that showed an improvement in catalytic efficiency (Vmax/KM) of more than 150 times that of wild-type PaS. The substrate scope of PaS was expanded and a modest increase in thermostability was observed. Finally, molecular dynamics simulations of enzyme-substrate complexes were used to provide qualitative insight into the structural origin of the observed effects.The authors thank the World Anti-Doping Agency’s Science Research Grants (13A13MM and 16A06MM), the Swedish Research Council (VR, Grant 2015-04928), as well as the Knut and Alice Wallenberg and Wenner-Gren foundations for financial support as well as fellowships to SCLK and AP respectively. All computational work in this paper was supported by computational resources provided by the Swedish National Infrastructure for Computing (SNIC, grants 2016-34-27 and 2017-12-11)

Representative structures from RMSD-clustering of MD trajectories.

PDB files contain representative structures of RMSD-based clusters obtained from the simulation of each variant using the combined last 50ns of all three replicas of the simulation perfomed for each variant. The clustering was performed on all heavy atoms of the side chains of the following residues: 13, 14, 50, 51, 72, 73, 74, 75, 113, 115, 138, 139, 155, 156, 157, 158, 159, 160, 211, 212, 317, 318, 321, 325, 330, 331, 375, as well as heavy atoms of the substrate. In each case the structures in each trajectory were aligned using backbone heavy atoms of the entire protein prior to clustering. Clustering was performed with cpptraj (Amber Tools) using average linkage algorithm. Summary of the clustering for each system can be found in *summary.out files, which contain the list of all clusters determined through the clustering analysis, with number of frames in each cluster, total fraction of the simulation these frames consittute, average distance between points in the cluster (AvgDist), standard deviation of points in the cluster (Stdev), centroid frame (Centroid) and average distance of a given cluster to every other cluster (AvgCDist). PDB files containing the representative structures of each cluster are labelled with the number corresponding to the number of a given cluster as listed in *summary.out file

Epiandrosterone sulfate prolongs the detectability of testosterone, 4-androstenedione, and dihydrotestosterone misuse by means of carbon isotope ratio mass spectrometry

In the course of investigations into the metabolism of testosterone (T) by means of deuterated T and hydrogen isotope ratio mass spectrometry, a pronounced influence of the oral administration of T on sulfoconjugated steroid metabolites was observed. Especially in case of epiandrosterone sulfate (EPIA_S), the contribution of exogenous T to the urinary metabolite was traceable up to 8 days after a single oral dose of 40 mg of T. These findings initiated follow-up studies on the capability of EPIA_S to extend the detection of T and T analogue misuse by carbon isotope ratio (CIR) mass spectrometry in sports drug testing. Excretion study urine samples obtained after transdermal application of T and after oral administration of 4-androstenedione, dihydrotestosterone, and EPIA were investigated regarding urinary concentrations and CIR. With each administered steroid, EPIA_S was significantly depleted and prolonged the detectability when compared to routinely used steroidal target compounds by a factor of 2 to 5. In order to simplify the sample preparation procedure for sulfoconjugated compounds, enzymatic cleavage by Pseudomonas aeruginosa arylsulfatase was tested and implemented into CIR measurements for the first time. Further simplification was achieved by employing multidimensional gas chromatography to ensure the required peak purity for CIR determinations, instead of sample purification strategies using liquid chromatographic fractionation. Taking into account these results that demonstrate the unique and broad applicability of EPIA_S for the detection of illicit administrations of T or T-related steroids, careful consideration of how this steroid can be implemented into routine doping control analysis appears warranted