Enhancing the Steroid Sulfatase Activity of the Arylsulfatase from Pseudomonas aeruginosa

Abstract

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 chromatographymass 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 enzymesubstrate complexes were used to provide qualitative insight into the structural origin of the observed effects.

Profiling Urinary Sulfate Metabolites With Mass Spectrometry

“…Within the PaS enzyme classes, there was also a small observed increase in the hydrolysis of saturated sulfates (m/z 97) from the WT-PaS to the LEF-PaS mutant. This trend aligned with the aims of the previous directed evolution study, which sought to improve the catalytic efficiency of the PaS enzyme for the hydrolysis of the saturated alkyl sulfate ester testosterone sulfate (Uduwela et al, 2018).…”

“…Glucuronylation, performed by uridine 5′-diphospho-glucuronosyltransferases (UGTs) has traditionally been the main focus of these investigations, as the major excretory phase II pathway, and due to the ready availability of β-glucuronidase enzymes for selective deconjugation. More recently, the study of sulfate conjugates has gained prominence due to the intriguing interplay between sulfotransferase (SULT) mediated synthesis, sulfatase promoted hydrolysis and a range of transport phenomena ( Foster and Mueller, 2018 ; Uduwela et al, 2018 ; Günal et al, 2019 ). The systematic study of the sulfated metabolome in a variety of contexts will be pivotal in revealing the roles of sulfation pathways in biology.…”

Gut Microbiota Exceeds Cervical Microbiota for Early Diagnosis of Endometriosis

“…Taking into account the aforementioned specificities of SCFAs, we hypothesize that the reduction of L. Ruminococcus may result in decreased concentration of protective metabolites in the human intestine, leading to the development of diseases. On the other hand, arylsulfatase from Pseudomonas aeruginosa was able to enhance the steroid sulfatase activity ( Uduwela et al., 2018 ), which is capable of catalyzing sulfated steroid precursors to the free steroid. Enhanced steroid sulfatase activity included catalyzing estrone sulfate to estrone, a weak estrogen.…”

Transition-State Interactions in a Promiscuous Enzyme: Sulfate and Phosphate Monoester Hydrolysis by Pseudomonas aeruginosa Arylsulfatase

“…[21][22][23][24][25][26][27][28][29][30][31][32][33][34][35][36][37] In addition computational simulations have been employed to pin-point transition state interactions. 27,28,[38][39][40][41][42][43] *We thank Marko Hyvonen for his help in the preparation of structural figures.…”

“…Here ,and in the work of Luo et al, 38 residues that -in our analysis -are assigned only indirect roles affecting the nature of the TS and leaving group stabilization (K113, H115, fGly51) feature more prominently. A third study 43 uses molecular dynamics simulations with active site group ground state pKa values validated by PROPKA to define a model that features K375, but not His211.…”