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 k cat and k cat/K m 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 k cat 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. However, none of the mutants screened possessed a significant activity against either of the β-sialosides.