Mutants provide evidence of the importance of glycosydic chains in the activation of lipase 1 from Candida rugosa.
Sequence analysis of Candida rugosa lipase 1 (LIP1) predicts the presence of three N-linked glycosylation sites at asparagine 291, 314, 351. To investigate the relevance of sugar chains in the activation and stabilization of LIP1, we directed site mutagenesis to replace the above mentioned asparagine with glutamine residues. Comparison of the activity of mutants with that of the wild-type (wt) lipase indicates that both 314 and 351 Asn to Gln substitutions influence, although at a different extent, the enzyme activity both in hydrolysis and esterification reactions, but they do not alter the enzyme water activity profiles in organic solvents or temperature stability. Introduction of Gln to replace Asn351 is likely to disrupt a stabilizing interaction between the sugar chain and residues of the inner side of the lid in the enzyme active conformation. The effect of deglycosylation at position 314 is more difficult to explain and might suggest a more general role of the sugar moiety for the structural stability of lipase 1. Conversely, Asn291Gln substitution does not affect the lipolytic or the esterase activity of the mutant that behaves essentially as the wt enzyme. This observation supports the hypothesis that changes in activity of Asn314Gln and Asn351Gln mutants are specifically due to deglycosylation.
Brocca
S
S
Department of Biotechnology and Biosciences, University of Milano-Bicocca, Milano, Italy.
Persson
M
M
Wehtje
E
E
Adlercreutz
P
P
Alberghina
L
L
Lotti
M
M
D001216:Asparagine / Q000737:chemistry; D015153:Blotting, Western; D002175:Candida / Q000201:enzymology*; D004789:Enzyme Activation; D005973:Glutamine / Q000737:chemistry; D006027:Glycosides / Q000737:chemistry*; D006031:Glycosylation; D006863:Hydrogen-Ion Concentration; D008049:Lipase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D010843:Pichia / Q000201:enzymology; D010957:Plasmids / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013696:Temperature; D014867:Water / Q000378:metabolism
D006027:Glycosides; D011994:Recombinant Proteins; D014867:Water; D005973:Glutamine; D001216:Asparagine; D008049:Lipase