Part:BBa_K4395007

SpyTag-RsAs-SpyCatcher is a fusion protein whose thermostability is increased by the SpyTag-SpyCatcher tagging system while the catalytic activity not affected. RsAs (Rauvolfia serpentina Arbutin Synthase), a recently detected glucosyltransferase (GT), catalyzes the biosynthesis of arbutin. It is a novel member of the Class IV GTs belonging to the Nucleotide Recognition Domain type1β (NRD1β) family of glycosyltransferases. The transferase exhibits the broadest acceptor substrate specificity compared to any of the glucosyltransferases previously described [1]. In addition, this enzyme has been actively expressed in E.coli [2]. These characteristics of RsAs attract us and thus we choose it as one of the candidates UGTs of our experiment. The SpyTag/SpyCatcher system is a split-in-two of the immunoglobulin-like collagen adhesion domain of Streptococcus pyogenes denoted CnaB2. CnaB2 is characterized by an internal isopeptide bond between residue Lys31 and residue Asp117. When split in two, one of which containing Lys31 and another containing Asp117, they associate and spontaneously form the isopeptide bond, thus join together [3] The composite part SpyTag-RsAs-SpyCatcher is a fusion protein cyclized by the SpyTag/SpyCatcher system, whose two components are fused on both ends. This cyclization process increases the thermostability of the enzyme, enabling catalysis in an unfavored heated system [4].

References: [1] Hefner T, Arend J, Warzecha H, et al. Arbutin synthase, a novel member of the NRD1β glycosyltransferase family, is a unique multifunctional enzyme converting various natural products and xenobiotics[J]. Bioorganic & Medicinal Chemistry, 2002, 10(6):1731-1741. [2] Arend J , Warzecha H , Hefner T , et al. Utilizing genetically engineered bacteria to produce plant‐specific glucosides[J]. Biotechnology & Bioengineering, 2001, 76(2):126-31. [3] Long Li, Jacob O. Fierer, et al. Structural Analysis and Optimization of the Covalent Association between SpyCatcher and a Peptide Tag J Mol Biol. 2014 January 23; 426(2): 309–317 [4] Xiao-Bao Suna, Jia-Wen Caoa,b, et al. SpyTag/SpyCatcher molecular cyclization confers protein stability and resilience to aggregation