Change of Bacillus cereus Flavonoid O-Triglucosyltransferase Into Flavonoid O-Monoglucosyltransferase by Error-Prone Polymerase Chain Reaction

• Published in: Journal of microbiology and biotechnology Vol. 20; no. 10; pp. 1393 - 1396
• Main Authors: Jung, N.R., Konkuk University, Seoul, Republic of Korea, Joe, E.J., Konkuk University, Seoul, Republic of Korea, Kim, B.G., Konkuk University, Seoul, Republic of Korea, Ahn, B.C., Konkuk University, Seoul, Republic of Korea, Park, J.C., National Institute of Animal Science, RDA, Suwon, Republic of Korea, Chong, Y.H., Konkuk University, Seoul, Republic of Korea, Ahn, J.H., Konkuk University, Seoul, Republic of Korea
• Format: Journal Article
• Language: English
• Published: Seoul Korean Society for Applied Microbiology 01.10.2010; 한국미생물·생명공학회
• Subjects: Amino Acid Sequence; BACILLUS CEREUS; Bacillus cereus - chemistry; Bacillus cereus - enzymology; Bacillus cereus - genetics; Bacterial Proteins - chemistry; Bacterial Proteins - genetics; Bacterial Proteins - metabolism; Binding Sites; Biological and medical sciences; Biotechnology; FLAVONOIDE; FLAVONOIDES; FLAVONOIDS; Flavonoids - metabolism; Fundamental and applied biological sciences. Psychology; GLICOSILTRANSFERASAS; Glycosylation; GLYCOSYLTRANSFERASE; GLYCOSYLTRANSFERASES; Glycosyltransferases - chemistry; Glycosyltransferases - genetics; Glycosyltransferases - metabolism; Models, Molecular; Molecular Sequence Data; Polymerase Chain Reaction; Protein Engineering; Protein Structure, Secondary; Substrate Specificity; 생물학; Polymerase chain reaction; Bacillus cereus; Bacillales; Enzyme; Transferases; glycosyltransferase; Glycosyltransferases; Bacteria; Bacillaceae; Flavonoid
• ISSN: 1017-7825; 1738-8872
• DOI: 10.4014/jmb.1003.03005

The attachment of sugar to flavonoids enhances their solubility. Glycosylation is performed primarily by uridine diphosphate-dependent glycosyltransferases (UGTs). The UGT from Bacillus cereus, BcGT-1, transferred three glucose molecules into kaempferol. The structural analysis of BcGT-1 showed that its substrate binding site is wider than that of plant flavonoid monoglucosyltransferases. In order to create monoglucosyltransferase from BcGT-1, the error-prone polymerase chain reaction (PCR) was performed. We analyzed 150 clones. Among them, two mutants generated only kaempferol O-monoglucoside, albeit with reduced reactivity. Unexpectedly, the two mutants harbored mutations in the amino acids located outside of the active sites. Based on the modeled structure of BcGT-1, it was proposed that the local change in the secondary structure of BcGT-1 caused the alteration of triglucosyltransferase into monoglucosyltransferase.