期刊
PROTEIN ENGINEERING DESIGN & SELECTION
卷 18, 期 5, 页码 239-246出版社
OXFORD UNIV PRESS
DOI: 10.1093/protein/gzi027
关键词
N-acetylneuraminic acid lyase; sialic acid mimetics; structure-guided saturation mutagenesis; synthesis
资金
- Engineering and Physical Sciences Research Council [GR/S99419/01] Funding Source: researchfish
Analogues of N-acetylneuraminic acid (sialic acid, NANA, Neu5Ac), including 6-dipropylcarboxamides, have been found to be selective and potent inhibitors of influenza sialidases. Sialic acid analogues are, however, difficult to synthesize by traditional chemical methods and the enzyme N-acetylneuraminic acid lyase (NAL) has previously been used for the synthesis of a number of analogues. The activity of this enzyme towards 6-dipropylcarboxamides is, however, low. Here, we used structure-guided saturation mutagenesis to produce variants of NAL with improved activity and specificity towards 6-dipropylcarboxamides. Three residues were targeted for mutagenesis, Asp191, Glu192 and Ser208. Only substitution at position 192 produced significant improvements in activity towards the dipropylamide. One variant, E192N, showed a 49-fold improvement in catalytic efficiency towards the target analogue and a 690-fold shift in specificity from sialic acid towards the analogue. These engineering efforts provide a scaffold for the further tailoring of NAL for the synthesis of sialic acid mimetics.
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