Neuraminidase Substrate Promiscuity Permits a Mutant Micromonospora viridifaciens Enzyme To Synthesize Artificial Carbohydrates

Mutation of the nucleophilic amino acid residue tyrosine to the small nonpolar residue glycine (Y370G) in the active site of Micromonospora viridifaciens neuraminidase (MvNA) produces an efficient catalyst for the transfer of N-acetylneuraminic acid from an artificial substrate (i.e., phenyl N-acetyl-β-d-neuraminide) to a sugar acceptor (e.g., d-lactose, d-glucose, d-mannose, d-raffinose, d-allose, or d-fructose) to give N-acetyl-α-neuraminide coupled carbohydrate products. In addition, this mutant enzyme (MvNA Y370G) catalyzes the transfer of a sugar residue from the artificial substrate 2-fluorophenyl N-acetyl-β-d-neuraminide to methyl glycopyranoside acceptors. Interestingly, when trans-glycosylation reactions are conducted in aqueous solutions containing 30% (v/v) acetonitrile, the α-anomeric acceptors of methyl glucopyranoside and galactopyranoside generate higher product yields than do their corresponding β-anomers. Specifically, a 64 h reaction with 2-fluorophenyl N-acetyl-β-d-neuraminide as the li...