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
