Expanding substrate specificity of GT-B fold glycosyltransferase via domain swapping and high-throughput screening.
Glycosyltransferases (GTs) are crucial enzymes in the biosynthesis and diversification of therapeutically important natural products, and the majority of them belong to the GT-B superfamily, which is composed of separate N- and C-domains that are responsible for the recognition of the sugar acceptor and donor, respectively. In an effort to expand the substrate specificity of GT, a chimeric library with different crossover points was constructed between the N-terminal fragments of kanamycin GT (kanF) and the C-terminal fragments of vancomycin GT (gtfE) genes by incremental truncation method. A plate-based pH color assay was newly developed for the selection of functional domain-swapped GTs, and a mutant (HMT31) with a crossover point (N-kanF-669 bp and 753 bp-gtfE-C) for domain swapping was screened. The most active mutant HMT31 (50 kDa) efficiently catalyzed 2-DOS (aglycone substrate for KanF) glucosylation using dTDP-glucose (glycone substrate for GtfE) with k(cat)/K(m) of 162.8 +/- 0.1 mM(-1) min(-1). Moreover, HMT31 showed improved substrate specificity toward seven more NDP-sugars. This study presents a domain swapping method as a potential means to glycorandomization toward various syntheses of 2-DOS-based aminoglycoside derivatives.
Park
Sung-Hee
SH
Institute of Molecular Biology and Genetics, Interdisciplinary Program for Bioengineering, Seoul National University, Sillim-dong, Gwanak-gu, Seoul 151-742, South Korea.
Park
Hyung-Yeon
HY
Sohng
Jae Kyung
JK
Lee
Hee Chan
HC
Liou
Kwangkyoung
K
Yoon
Yeo Joon
YJ
Kim
Byung-Gee
BG
D000900:Anti-Bacterial Agents / Q000378:metabolism; D050260:Carbohydrate Metabolism; D019020:Directed Molecular Evolution; D016695:Glycosyltransferases / Q000235:genetics* / Q000378:metabolism*; D007700:Kinetics; D017434:Protein Structure, Tertiary / Q000235:genetics; D011995:Recombination, Genetic; D013379:Substrate Specificity
D000900:Anti-Bacterial Agents; D016695:Glycosyltransferases