DOI: 10.5061/dryad.p5n151t
title: Data from: Enhancing the steroid sulfatase activity of the arylsulfatase from Pseudomonas aeruginosa

description: 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 chromatography-mass 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
 enzyme-substrate complexes were used to provide qualitative insight into
 the structural origin of the observed effects.
description: Representative
 structures from RMSD-clustering of MD trajectories.PDB files contain
 representative structures of RMSD-based clusters obtained from the
 simulation of each variant using the combined last 50ns of all three
 replicas of the simulation perfomed for each variant. The clustering was
 performed on all heavy atoms of the side chains of the following residues:
 13, 14, 50, 51, 72, 73, 74, 75, 113, 115, 138, 139, 155, 156, 157, 158,
 159, 160, 211, 212, 317, 318, 321, 325, 330, 331, 375, as well as heavy
 atoms of the substrate. In each case the structures in each trajectory
 were aligned using backbone heavy atoms of the entire protein prior to
 clustering. Clustering was performed with cpptraj (Amber Tools) using
 average linkage algorithm. Summary of the clustering for each system can
 be found in *summary.out files, which contain the list of all clusters
 determined through the clustering analysis, with number of frames in each
 cluster, total fraction of the simulation these frames consittute, average
 distance between points in the cluster (AvgDist), standard deviation of
 points in the cluster (Stdev), centroid frame (Centroid) and average
 distance of a given cluster to every other cluster (AvgCDist). PDB files
 containing the representative structures of each cluster are labelled with
 the number corresponding to the number of a given cluster as listed in
 *summary.out file.Structures.zip