PMID- 25739756 OWN - NLM STAT- MEDLINE DCOM- 20160722 LR - 20151008 IS - 1942-7611 (Electronic) IS - 1942-7603 (Linking) VI - 7 IP - 10 DP - 2015 Oct TI - Pseudomonas aeruginosa arylsulfatase: a purified enzyme for the mild hydrolysis of steroid sulfates. PG - 903-11 LID - 10.1002/dta.1782 [doi] AB - 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 5alpha-androstane-3beta,17beta-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. CI - Copyright (c) 2015 John Wiley & Sons, Ltd. FAU - Stevenson, Bradley J AU - Stevenson BJ AD - Research School of Chemistry, Australian National University, Canberra, ACT, 2601, Australia. FAU - Waller, Christopher C AU - Waller CC AD - Research School of Chemistry, Australian National University, Canberra, ACT, 2601, Australia. FAU - Ma, Paul AU - Ma P AD - Research School of Chemistry, Australian National University, Canberra, ACT, 2601, Australia. FAU - Li, Kunkun AU - Li K AD - Research School of Chemistry, Australian National University, Canberra, ACT, 2601, Australia. FAU - Cawley, Adam T AU - Cawley AT AD - Racing New South Wales - Australian Racing Forensic Laboratory, Sydney, NSW, 1465, Australia. FAU - Ollis, David L AU - Ollis DL AD - Research School of Chemistry, Australian National University, Canberra, ACT, 2601, Australia. FAU - McLeod, Malcolm D AU - McLeod MD AUID- ORCID: 0000-0002-2343-3226 AD - Research School of Chemistry, Australian National University, Canberra, ACT, 2601, Australia. LA - eng PT - Journal Article PT - Research Support, Non-U.S. Gov't DEP - 20150305 PL - England TA - Drug Test Anal JT - Drug testing and analysis JID - 101483449 RN - 0 (Steroids) RN - 0 (Sulfates) RN - EC 3.1.6.1 (Arylsulfatases) SB - IM MH - Arylsulfatases/*metabolism MH - Enzyme Assays/methods MH - Gas Chromatography-Mass Spectrometry/methods MH - Humans MH - Hydrolysis MH - Pseudomonas aeruginosa/*enzymology MH - Steroids/analysis/*metabolism/urine MH - Sulfates/analysis/*metabolism/urine OTO - NOTNLM OT - chemoenzymatic synthesis OT - glucuronide OT - sports drug testing OT - steroid OT - sulfatase OT - sulfate ester EDAT- 2015/03/06 06:00 MHDA- 2016/07/23 06:00 CRDT- 2015/03/06 06:00 PHST- 2014/12/04 00:00 [received] PHST- 2015/02/04 00:00 [revised] PHST- 2015/02/04 00:00 [accepted] PHST- 2015/03/06 06:00 [entrez] PHST- 2015/03/06 06:00 [pubmed] PHST- 2016/07/23 06:00 [medline] AID - 10.1002/dta.1782 [doi] PST - ppublish SO - Drug Test Anal. 2015 Oct;7(10):903-11. doi: 10.1002/dta.1782. Epub 2015 Mar 5.