Structure-guided saturation mutagenesis of N-acetylneuraminic acid lyase for the synthesis of sialic acid mimetics.
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.
Williams
G J
GJ
Astbury Centre for Structural Biology, School of Biochemistry, University of Leeds, Leeds LS2 9JT, UK.
Woodhall
T
T
Nelson
A
A
Berry
A
A
D000577:Amides / Q000378:metabolism; D019943:Amino Acid Substitution; D032701:Biomimetics; D004926:Escherichia coli / Q000201:enzymology*; D015394:Molecular Structure; D016296:Mutagenesis; D019158:N-Acetylneuraminic Acid / Q000378:metabolism*; D007652:Oxo-Acid-Lyases / Q000737:chemistry* / Q000235:genetics*; D015202:Protein Engineering; D013329:Structure-Activity Relationship; D013379:Substrate Specificity
D000577:Amides; D007652:Oxo-Acid-Lyases; C018728:N-acetylneuraminate lyase; D019158:N-Acetylneuraminic Acid