期刊
BIOTECHNOLOGY AND BIOENGINEERING
卷 102, 期 4, 页码 988-994出版社
WILEY
DOI: 10.1002/bit.22150
关键词
natural product; antibiotics; glycosyltransferase; molecular evolution; domain swapping; high-throughput screening (HTS)
资金
- Ministry of Commerce, Industry and Energy [10023194]
- BioGreen 21 Program, Rural Development Administration, Republic of Korea. [20050401034682]
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 hip and 753 bp-gtjE-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.
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