Modification of an Arylsulfatase from <em>Pseudomonas</em> <em>aeruginosa</em> for anti-doping — ASN Events
  1. Schedule
  2. Session Ten - Poster Session C including Refreshments
  3. Modification of an Arylsulfatase from Pseudomonas aeruginosa for anti-doping

Modification of an Arylsulfatase from Pseudomonas aeruginosa for anti-doping (#314)

Dimanthi Uduwela 1 , Bradley Stevenson 1 , Malcolm McLeod 1
  1. Research School of Chemistry, Australian National University, Acton, ACT, 2601, Australia

Steroid abuse in sports is a timely concern and has received increased attention in recent years. Detection of a complete steroid profile is thus important to maintain fair play in sports. Steroid sulfates, are common phase II metabolites of steroids that remain undetected in anti-doping screens due to the unavailability of a suitable enzymatic or chemical method for deconjugation prior to GC/MS analysis(1).

An aryl sulfatase from Pseudomonas aeruginosa (PaS) has been found to show promiscuous activity in the hydrolysis of steroid sulfates(2). However, this enzyme requires enhanced activity to be useful in anti-doping context. This can be achieved by applying rational design to mutate amino acid residues neighboring the substrate binding pocket, to open it up more. Site saturation mutagenesis was performed on five such residues selected by analyzing the reported crystal structure of PaS(3). This resulted in three improved mutants from two sites. Hybridizing these beneficial mutants to produce double mutants, further improved activity towards testosterone sulfate. Kinetics were studied for the purified mutants and the best mutant showed a ~6 fold improvement, of catalytic efficiency,than wild type.

  1. Gomes, R.L. et al., J. Pharm. Biomed. Anal., 2009, 49(5), 1133-1140.
  2. Beil, S. et al., Eur. J. Biochem., 1995, 229(2), 385-394.
  3. Boltes, I. et al., Structure, 2001. 9(6), 483-491.