Characterization and engineering of glycosyltransferases responsible for steroid saponin biosynthesis in Solanaceous plants.
Solanaceous plants contain steroid saponins that have diverse biological and pharmacological activities. The structures of their sugar chains play an important role in their activities. A functional glucosyltransferase SaGT4A from Solanum aculeatissimum glucosylates both steroidal sapogenins and steroidal alkaloids. A potato (S. tuberosum) glycosyltransferase StSGT, which has a high degree of sequence homology with SaGT4A, exhibits the same substrate specificity toward steroidal compounds as SaGT4A. To identify the residues or domain structures responsible for these enzymatic activities, we determined the residues that are essential for SaGT4A activity, compared the specific activities of SaGT4A and StSGT, and constructed several SaGT4A/StSGT chimeric proteins, focusing on the donor-sugar recognition domain. These proteins were heterogeneously expressed in E. coli and purified, and their glycosyltransferase activities were evaluated using a coupled assay. His369 and Glu377, located in the consensus motif for plant glycosyltransferases, and Cys121, Cys247, and Cys370 were shown to be important for SaGT4A activity. StSGT exhibited more activity with UDP-galactose as a sugar donor than with UDP-glucose, whereas SaGT4A exhibited glucosyltransferase activity exclusively. The sugar selectivities of SaGT4A and StSGT were not altered by exchanging their domains, and some of the chimeric proteins showed no activity. These results suggest that the differences in the SaGT4A and StSGT amino acid sequences do not simply reflect their distinct sugar-donor specificities. We also successfully converted the non-functional SaGT4A homolog, SaGT4R, into an active glucosyltransferase.
Kohara
Atsuko
A
RIKEN Plant Science Center, Tsurumi, Yokohama, Kanagawa 230-0045, Japan.
Nakajima
Chiharu
C
Yoshida
Shigeo
S
Muranaka
Toshiya
T
D000595:Amino Acid Sequence; D017124:Conserved Sequence; D005818:Genetic Engineering; D016695:Glycosyltransferases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D050506:Mutant Chimeric Proteins / Q000737:chemistry / Q000378:metabolism; D010940:Plant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012503:Saponins / Q000096:biosynthesis*; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D019657:Solanaceae / Q000201:enzymology* / Q000235:genetics; D011198:Solanum tuberosum / Q000201:enzymology; D013256:Steroids / Q000096:biosynthesis*
D050506:Mutant Chimeric Proteins; D010940:Plant Proteins; D011994:Recombinant Proteins; D012503:Saponins; D013256:Steroids; D016695:Glycosyltransferases