Understanding the sequence-structure-function relationship of the large arylsulfate sulfotransferase (ASST) enzyme family for engineering novel sulfation biocatalysts

硫酸生物分子广泛存在于自然界中，在生物功能中发挥着重要作用。在负责硫酸酸化的酶中，芳基硫酸盐硫转移酶（ASSTs）是一种有趣的生物催化剂，因为它们使用简单的芳香硫酸盐（如对硝基苯硫酸盐）作为供体，而依赖PAPS的硫转移酶则使用复杂且不太稳定的PAPS作为供体。然而，对ASSTs知之甚少（仅描述了一种3d结构及其分子机制，根据其生物化学或基因组背景暂定划分为不同的类别，仅确定了一种天然供体和一种受体底物）。根据我们对2244个ASSTs基因序列的初步系统发育分析，我们确定了19个进化支具有合理的引导陷阱值。与CAZY或Sulfatlas数据库类似，实际的19个分支中的每一个都对应于不同的底物特异性或/和机制。然而，由于生物化学和结构数据稀缺，这一假设不能被今天的实验数据所挑战。此外，许多分支（枝）与分类学一致，这就提出了一个明显的问题，即底物特异性可能是与分类学相关的一个特征。在SulfASST项目中，我们结合了生物信息学、生物化学、酶学、结构生物学、分子建模和蛋白质工程等方面的互补方法，获得了有关助理助理酶的大量信息。在初步的系统发育分析的基础上，将对19个亚家族（分支）中的每个亚家族（分支）进行表达和筛选供体和受体底物。6-8个经过精心挑选的ASSTs代表的酶晶体学应该提供催化和选择性分子方面的宝贵细节（底物，区域化学）。定向酶进化（KnowVolution）和建模将允许为生物技术目的获得量身定制的生物催化剂。最后，对ASSTs的深入表征和所得结果的合理化将使我们能够：确定底物特异性是否与系统发育相关；了解ASSTs基因的基因组背景是否指示底物或生物活性；解读底物特异性/混杂性和区域选择性的结构决定因素；确定酶机制是否在不同的亚科（分支）中保守；通过分子模型预测底物选择性和区域选择性。

Sulfated biomolecules are widespread in nature and play important roles in biological functions. Among the enzymes responsible for sulfation, ArylSulfate SulfoTransferases (ASSTs) are interesting biocatalysts as they use simple aromatic sulfates such as para-nitrophenyl sulfate as donors in comparison to PAPS-dependent sulfotransferases that use the complex and less stable PAPS as donor. However, very few is known about ASSTs (only one 3D-structure and its molecular mechanism described, tentative assignment into different classes according to their biochemistry or genomic context, only one natural donor and one acceptor substrate identified). According to our preliminary phylogenetic analysis on 2244 sequences of ASSTs genes, we identified 19 clades displaying reasonable boots-trap values. In analogy to CAZY or Sulfatlas databases, each of the actual 19 clades could correspond to a varying substrate specificity or/and mechanism. However, since biochemical and structural data are scarce, this hypothesis cannot be challenged by experimental data today. Moreover many of the branches (clades) coincide with taxonomy, which raises the obvious question that substrate specificity might be a trait which is linked to taxonomy.In the SulfASST project, we uses a combination of complementary approaches in bioinformatics, biochemistry, enzymology, structural biology, molecular modeling and protein engineering to obtain substantial information on the ASST enzymes. Based on the preliminary phylogenetic analysis, one representative of each of the 19 subfamilies (clades) will be expressed and screened for donor and acceptor substrates. Enzyme crystallography of 6-8 soundly selected representatives of ASSTs should provide precious details on molecular aspects of catalysis and selectivity (substrate, regiochemistry). Directed enzyme evolution (KnowVolution) and modeling will allow to obtain tailor-made biocatalysts for biotechnological purposes. Finally, this in-depth characterization of the ASSTs and rationalization of the obtained results will enable to: determine if substrate specificity is correlated to phylogeny; know if the genomic context of ASSTs genes is indicative of substrate or biological activity; decipher the structural determinants of substrate specificity/promiscuity and regioselectivity; define if enzyme mechanism is conserved throughout the different subfamilies (clades); predict substrate selectivity and regioselectivity by molecular modeling.