Pseudomonas aeruginosa arylsulfatase: a purified enzyme for the mild hydrolysis of steroid sulfates.
The hydrolysis of sulfate ester conjugates is frequently required prior to analysis for a range of analytical techniques including gas chromatography-mass spectrometry (GC-MS). Sulfate hydrolysis may be achieved with commercial crude arylsulfatase enzyme preparations such as that derived from Helix pomatia but these contain additional enzyme activities such as glucuronidase, oxidase, and reductase that make them unsuitable for many analytical applications. Strong acid can also be used to hydrolyze sulfate esters but this can lead to analyte degradation or increased matrix interference. In this work, the heterologously expressed and purified arylsulfatase from Pseudomonas aeruginosa is shown to promote the mild enzyme-catalyzed hydrolysis of a range of steroid sulfates. The substrate scope of this P. aeruginosa arylsulfatase hydrolysis is compared with commercial crude enzyme preparations such as that derived from H. pomatia. A detailed kinetic comparison is reported for selected examples. Hydrolysis in a urine matrix is demonstrated for dehydroepiandrosterone 3-sulfate and epiandrosterone 3-sulfate. The purified P. aeruginosa arylsulfatase contains only sulfatase activity allowing for the selective hydrolysis of sulfate esters in the presence of glucuronide conjugates as demonstrated in the short three-step chemoenzymatic synthesis of 5α-androstane-3β,17β-diol 17-glucuronide (ADG, 1) from epiandrosterone 3-sulfate. The P. aeruginosa arylsulfatase is readily expressed and purified (0.9 g per L of culture) and thus provides a new and selective method for the hydrolysis of steroid sulfate esters in analytical sample preparation.
Research School of Chemistry, Australian National University, Canberra, ACT, 2601, Australia.
Bradley J
BJ
Stevenson
Research School of Chemistry, Australian National University, Canberra, ACT, 2601, Australia.
Christopher C
CC
Waller
Research School of Chemistry, Australian National University, Canberra, ACT, 2601, Australia.
Paul
P
Ma
Research School of Chemistry, Australian National University, Canberra, ACT, 2601, Australia.
Kunkun
K
Li
Racing New South Wales - Australian Racing Forensic Laboratory, Sydney, NSW, 1465, Australia.
Adam T
AT
Cawley
Research School of Chemistry, Australian National University, Canberra, ACT, 2601, Australia.
David L
DL
Ollis
Research School of Chemistry, Australian National University, Canberra, ACT, 2601, Australia.
Malcolm D
http://orcid.org/0000-0002-2343-3226
MD
McLeod
D001192:Arylsulfatases / Q000378:metabolism*; D057075:Enzyme Assays / Q000379:methods; D008401:Gas Chromatography-Mass Spectrometry / Q000379:methods; D006801:Humans; D006868:Hydrolysis; D011550:Pseudomonas aeruginosa / Q000201:enzymology*; D013256:Steroids / Q000032:analysis / Q000378:metabolism* / Q000652:urine; D013431:Sulfates / Q000032:analysis / Q000378:metabolism* / Q000652:urine
D013256:Steroids; D013431:Sulfates; D001192:Arylsulfatases
chemoenzymatic synthesis; glucuronide; sports drug testing; steroid; sulfatase; sulfate ester