Data from: Enhancing the steroid sulfatase activity of the arylsulfatase from Pseudomonas aeruginosa

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.

甾体硫酸酯（steroidal sulfate esters）在众多生理过程中发挥核心作用，可作为内源性性激素的储备库，同时在甾体代谢物库中占据重要比例。因此，对甾体硫酸盐的分析在医学、运动药物检测等领域至关重要。尽管可通过液相色谱-质谱联用（liquid chromatography-mass spectrometry）技术直接检测甾体硫酸盐，但包括气相色谱-质谱联用（gas chromatography-mass spectrometry）在内的诸多分析方法，却因缺乏适用于分析前步骤的硫酸酯水解酶法或化学法而受限。优化甾体硫酸酯水解的方法，将为这类广泛存在的代谢物的研究拓展分析路径。铜绿假单胞菌来源的芳基硫酸酯酶（arylsulfatase from Pseudomonas aeruginosa，PaS）是一种可水解甾体硫酸盐的纯化酶，但该酶的水解活性与底物谱均有待优化，才能满足分析应用的需求。本研究通过半理性设计（semi-rational design）技术，对酶活性位点邻近的氨基酸残基开展诱变，分别针对单残基位点与多残基位点实施诱变操作。通过超高效液相色谱-质谱联用（ultra-high performance liquid chromatography-mass spectrometry，UPLC-MS）技术对突变体库进行筛选，以检测其对硫酸睾酮的甾体硫酸盐水解活性。该研究明确了甾体硫酸盐的结合口袋，并获得了3株催化效率（Vmax/KM）较野生型PaS提升150倍以上的突变体。PaS的底物谱得到拓展，同时其热稳定性也出现小幅提升。最后，本研究通过酶-底物复合物的分子动力学模拟（molecular dynamics simulations），对观测到的性能变化的结构根源进行了定性解析。