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Expanding substrate specificity of GT-B fold glycosyltransferase via domain swapping and high-throughput screening

Title
Expanding substrate specificity of GT-B fold glycosyltransferase via domain swapping and high-throughput screening
Authors
Park S.-H.Park H.-Y.Sohng J.K.Lee H.C.Liou K.Yoon Y.J.Kim B.-G.
Ewha Authors
윤여준
SCOPUS Author ID
윤여준scopus
Issue Date
2009
Journal Title
Biotechnology and Bioengineering
ISSN
0006-3592JCR Link
Citation
vol. 102, no. 4, pp. 988 - 994
Indexed
SCI; SCIE; SCOPUS WOS scopus
Abstract
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 glyco- randomization toward various syntheses of 2-DOS-based aminoglycoside derivatives. © 2008 Wiley Periodicals, Inc.
DOI
10.1002/bit.22150
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자연과학대학 > 화학·나노과학전공 > Journal papers
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