The hydrolase and transferase activity of an inverting mutant sialidase using non-natural beta-sialoside substrates.
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.
Watson
Jacqueline N
JN
Department of Chemistry, Simon Fraser University, 8888 University Drive, Burnaby, British Columbia, V5A 1S6, Canada.
Indurugalla
Deepani
D
Cheng
Lydia L
LL
Narine
Arun A
AA
Bennet
Andrew J
AJ
D004591:Electrophoresis, Polyacrylamide Gel; D006031:Glycosylation; D006861:Hydrogen Peroxide / Q000378:metabolism; D006867:Hydrolases / Q000378:metabolism*; D007700:Kinetics; D008847:Micromonospora / Q000201:enzymology; D008958:Models, Molecular; D009439:Neuraminidase / Q000235:genetics / Q000378:metabolism*; D019906:Nuclear Magnetic Resonance, Biomolecular; D010100:Oxygen / Q000378:metabolism; D012794:Sialic Acids / Q000378:metabolism*; D013379:Substrate Specificity; D014166:Transferases / Q000378:metabolism*
D012794:Sialic Acids; C515496:phenyl beta-sialoside; D006861:Hydrogen Peroxide; D014166:Transferases; D006867:Hydrolases; D009439:Neuraminidase; D010100:Oxygen