The hydrolase and transferase activity of an inverting mutant sialidase using non-natural β-sialoside substrates

The Y370G inverting mutant sialidase from Micromonospora viridifaciens possesses β-sialidase activity with phenyl β-sialoside (Ph-βNeuAc) to give α-sialic acid as the first formed product. The derived catalytic rate constants for kcat and kcat/Km are 13.3 ± 0.3 and (2.9 ± 0.3) × 105 M-1 s-1, respectively. This enzyme is highly specific for the phenyl substrate, with substituted phenyl and thiophenyl leaving groups having kcat values that are at least 1000-fold lower. In addition, the Y370G mutant can transfer the sialic acid moiety from Ph-βNeuAc to lactose in yields of up to 13%. Greater than 90% of the sialyl-lactose product formed in the coupling reactions is the α-2,6-isomer. A library encoding 6 × 105 different sialidases was constructed by mutating Y370, E260, T309, N310, and N311, residues that include and are proximal the catalytic tyrosine residue. A total of 2628 individuals were screened for hydrolytic activity against 4-nitrophenyl 2-thio-β-sialoside and 4-methylumbelliferyl β-sialoside. Howev...