期刊
ANGEWANDTE CHEMIE-INTERNATIONAL EDITION
卷 57, 期 32, 页码 10153-10157出版社
WILEY-V C H VERLAG GMBH
DOI: 10.1002/anie.201804831
关键词
biocatalysis; fructose-6-phosphate aldolase; HPTLC screening; protein engineering; stereoselectivity
资金
- European Union's Horizon 2020 research and innovation program [635595]
- Spanish Ministerio de Economia y Competitividad (MINECO)
- Fondo Europeo de Desarrollo Regional (FEDER) [CTQ2015-63563-R]
A structure-guided engineering of fructose-6-phosphate aldolase was performed to expand its substrate promiscuity toward aliphatic nucleophiles, that is, unsubstituted alkanones and alkanals. A smart combinatorial library was created targeting residues D6, T26, and N28, which form a binding pocket around the nucleophilic carbon atom. Double-selectivity screening was executed by high-performance TLC that allowed simultaneous determination of total activity as well as a preference for acetone versus propanal as competing nucleophiles. D6 turned out to be the key residue that enabled activity with non-hydroxylated nucleophiles. Altogether 25 single- and double-site variants (D6X and D6X/T26X) were discovered that show useful synthetic activity and a varying preference for ketone or aldehyde as the aldol nucleophiles. Remarkably, all of the novel variants had completely lost their native activity for cleavage of fructose 6-phosphate.
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