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PEDS Advance Access published online on May 16, 2005
Protein Engineering Design and Selection, doi:10.1093/protein/gzi027
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© The Author 2005. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oupjournals.org
Received April 11, 2005
Accepted April 12, 2005

Article

Structure-guided saturation mutagenesis of N-acetylneuraminic acid lyase for the synthesis of sialic acid mimetics

G.J. Williams 1, T. Woodhall 2, A. Nelson 2, and A. Berry 1*

1 Astbury Centre for Structural Biology, University of Leeds, Leeds LS2 9JT, UK; School of Biochemistry and Microbiology, University of Leeds, Leeds LS2 9JT, UK
2 Astbury Centre for Structural Biology, University of Leeds, Leeds LS2 9JT, UK; School of Chemistry, University of Leeds, Leeds LS2 9JT, UK

* To whom correspondence should be addressed.
A. Berry, E-mail: a.berry{at}leeds.ac.uk


  Abstract

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.

Keywords: N-acetylneuraminic acid lyase; sialic acid mimetics; structure-guided saturation mutagenesis; synthesis.
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