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PEDS Advance Access originally published online on May 16, 2005
Protein Engineering Design and Selection 2005 18(5):239-246; 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

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

G.J. Williams1,2,3, T. Woodhall1,4, A. Nelson1,4 and A. Berry1,2,5

1Astbury Centre for Structural Biology, 2School of Biochemistry and Microbiology and 4School of Chemistry, University of Leeds, Leeds LS2 9JT, UK 3Present address: School of Pharmacy, University of Wisconsin, 777 Highland Avenue, Madison, WI 53705, USA

5 To whom correspondence should be addressed. E-mail: a.berry{at}leeds.ac.uk

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

Received April 11, 2005; accepted April 12, 2005.

Edited by Tony Wilkinson


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