Reconstitution of antibiotics glycosylation by domain exchanged chimeric glycosyltransferase

Glycosyltransferase (GT), which catalyzes the transfer of nucleotide-activated sugar to aglycone, is the key enzyme for the preparation of a variety of glycosylated natural products. In this study, we report that the substrate specificity of GTs can be altered via domain exchanging accompanied by rationally designed peptide linkers. Comprehensive similarity searches were first conducted based upon the amino acid sequences of antibiotic GTs. The phylogenetic tree displays a distinct separation of 25 antibiotic GTs into three groups according to their aglycone substrates. Two different groups of GTs, KanF (aminoglycoside) and GtfE (glycopeptide) were employed for domain exchanging of GT-B fold enzyme. In order to determine linker peptide sequences, the secondary structures of KanF and GtfE were carefully evaluated via 3D-PSSM. The N-terminal domain of KanF and the C-terminal domain of GtfE were fused with rationally designed linker sequences. The hybrid GT, referred to as KE chimera, showed hybrid activity toward dTDP-d-glucose and 2-deoxystreptamine. Moreover, the key catalytic amino acid residues of KE chimera were investigated via protein modeling and docking simulations.