The Hydrolase and Transferase Activity of an Inverting Mutant Sialidase Using Non-natural β-Sialoside Substrates

Abstract

The Y370G inverting mutant sialidase from Micromonospora viridifaciens possesses beta-sialidase activity with phenyl beta-sialoside (Ph-betaNeuAc) to give alpha-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) x 10(5) 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-betaNeuAc to lactose in yields of up to 13%. Greater than 90% of the sialyl-lactose product formed in the coupling reactions is the alpha-2,6-isomer. A library encoding 6 x 10(5) 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-beta-sialoside and 4-methylumbelliferyl beta-sialoside. However, none of the mutants screened possessed a significant activity against either of the beta-sialosides.