RESUMEN
Glycosyltransferases (GT) catalyze the glycosylation of bioactive natural products, including peptides and proteins, flavonoids, and sterols, and have been extensively used as biocatalysts to generate glycosides. However, the often narrow substrate specificity of wild-type GTs requires engineering strategies to expand it. The GT-B structural family is constituted by GTs that share a highly conserved tertiary structure in which the sugar donor and acceptor substrates bind in dedicated domains. Here, we have used this selective binding feature to design an engineering process to generate chimeric glycosyltransferases that combine auto-assembled domains from two different GT-B enzymes. Our approach enabled the generation of a stable dimer with broader substrate promiscuity than the parent enzymes that were related to relaxed interactions between domains in the dimeric GT-B. Our findings provide a basis for the development of a novel class of heterodimeric GTs with improved substrate promiscuity for applications in biotechnology and natural product synthesis.
Asunto(s)
Biocatálisis , Glicosiltransferasas , Flavonoides/química , Glicosilación , Glicosiltransferasas/química , Glicosiltransferasas/genética , Especificidad por Sustrato , Dominios Proteicos , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Bioingeniería/métodosRESUMEN
Dictyoglomus thermophilum ß-d-xylosidase DtXyl is attractive as a potential thermostable biocatalyst able to produce biologically active ginsenosides intermediates from ß-(1,2)-D-xylosylated compounds, including Notoginsenoside-R1. DtXyl was expressed as an active N-terminal His-tagged protein, and its crystal structure was solved in presence or absence of d-xylose product. Modelling of notoginsenoside R1 in DtXyl active site led to the identification of several hydrophobic residues interacting in close contact to the substrate hydrophobic core. Unlike other residues involved in substrate binding, these residues are not conserved among GH39 xylosidase family, and their physico-chemical properties can be correlated to the efficient binding and subsequent hydrolysis of Notoginsenoside R1.