cs8b00376_si_001.pdf (5.64 MB)

Probing and Engineering Key Residues for Bis‑C‑glycosylation and Promiscuity of a C‑Glycosyltransferase

Download (5.64 MB)
journal contribution
posted on 23.04.2018, 00:00 by Dawei Chen, Shuai Fan, Ridao Chen, Kebo Xie, Sen Yin, Lili Sun, Jimei Liu, Lin Yang, Jianqiang Kong, Zhaoyong Yang, Jungui Dai
C-Glycosyltransferases (CGTs) are powerful tools for the C-glycosylation of natural and unnatural products. However, CGTs able to catalyze bis-C-glycosylation are very rare and the key amino acids of which have not been uncovered. Here, we discovered a C-glycosyltransferase MiCGTb from Mangifera indica that has the capacity for bis-C-glycosylation. Further studies on active-site motifs revealed that I152 of MiCGTb was the critical amino acid residue for the second C-glycosylation and its S60/V100/T104 residues were pivotal for bis-C-glycosylation activity. Moreover, we developed a panel of variants with acceptor and donor promiscuity by site-directed mutagenesis. Among these variants, a mutant MiCGT-E152L displayed a broader acceptor scope for bis-C-glycosylation, and three mutants of MiCGTb exhibited sugar donor promiscuity toward structurally varied α-d- and β-l-glycosyl donors. Our work provides insights into the pivotal amino acid residues of CGTs for bis-C-glycosylation and biocatalytic tools to efficiently produce structurally diverse bis-C-glycosides with two identical or different sugar moieties in drug discovery.

History

Exports