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 beenfound to be selective and potent inhibitors of influenzasialidases. Sialic acid analogues are, however, difficult tosynthesize bytraditional chemical methods and the enzymeN-acetylneuraminic acid lyase (NAL) has previously beenusedforthesynthesisofanumberofanalogues.Theactivityofthisenzymetowards6-dipropylcarboxamidesis,however,low.Here,weusedstructure-guidedsaturationmutagenesisto produce variants of NAL with improved activity andspecificitytowards6-dipropylcarboxamides.Threeresiduesweretargetedformutagenesis,Asp191,Glu192andSer208.Only substitution at position 192 produced significantimprovements in activity towards the dipropylamide. Onevariant, E192N, showed a 49-fold improvement in catalyticefficiency towards the target analogue and a 690-foldshift in specificity from sialic acid towards the analogue.These engineering efforts provide a scaffold for the furthertailoring of NAL for the synthesis of sialic acid mimetics.Keywords: N-acetylneuraminicacidlyase/sialicacidmimetics/structure-guided saturation mutagenesis/synthesisIntroductionN-Acetylneuraminicacid(sialicacid,NANA,Neu5Ac,3)isanessential component of complex carbohydrates, which playpivotal roles in recognition processes in a variety of importantcellular recognition processes (Schauer, 1982; Varki, 1993,1997; Schauer et al., 1995). These events are particularlywell exemplified in host–pathogen and host–parasite interac-tions, where the oligosaccharide is often required for invasion,infectivity and survival of the invading organism in the host.Sialic acid analogues therefore represent attractive targets fornovel chemotherapeutic agents against bacterial and viralinfections (Koketsu et al., 1997). For example, influenzavirus neuraminidase plays a crucial role in the life cycle ofthe virus and inhibitors of this enzyme such as zanamivir(GlaxoSmithKline’s Relenza