Neuraminidase substrate promiscuity permits a mutant Micromonospora viridifaciens enzyme to synthesize artificial carbohydrates.

Abstract

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 limiting reagent and the acceptors methyl α-d-galactopyranoside, methyl α-D-glucopyranoside, or methyl α-D-mannopyranoside gives trans-glycosylation product yields of 22%, 31%, or 34%, respectively. With methyl α-D-galactopyranoside as the acceptor, trans-glycosylations catalyzed by both MvNA Y370G and a 2,6-sialyltransferase yield identical products, which we identified as methyl N-acetyl-α-D-neuraminyl-(2 → 6)-α-D-galactopyranoside. The MvNA Y370G-catalyzed coupling of N-acetylneuraminic acid to these three methyl α-d-glycopyranoside acceptors is favored by factors of 18–27-fold over the competing hydrolysis reaction. These coupling efficiencies likely arise from nonselective interactions between the acceptor glycopyranoside and MvNA Y370G, which preferentially places a carbohydrate hydroxyl group rather than water in close proximity to the active site where this functionality intercepts the nascent neuraminyl oxacarbenium ion that is formed during cleavage of the glycosidic bond in the aryl N-acetyl-β-D-neuraminide donor. The ability to transfer N-acetylneuraminic acid from a stable and readily accessible donor to acceptor carbohydrates that are not substrates for sialyltransferases is one step on the path for the production of pseudohuman glycoproteins from nonmammalian cell lines.