Subject(s)
Acetonitriles/chemical synthesis , Benzaldehydes/chemistry , Benzaldehydes/chemical synthesis , Directed Molecular Evolution/methods , Acetonitriles/chemistry , Amino Acid Sequence , Base Sequence , Binding Sites , Catalysis , Lyases/chemistry , Lyases/genetics , Lyases/metabolism , Mutagenesis, Site-Directed , Pichia/genetics , Polymerase Chain Reaction , Prunus/enzymology , Prunus/genetics , Recombination, Genetic , Substrate SpecificityABSTRACT
(R)-2-chloromandelic acid represents a key pharmaceutical intermediate. Its production on large scale was hampered by low turnover rates and moderate enantiomeric excess (ee) using enzyme as well as metal catalysts. The cloning and heterologous overexpression of an (R)-hydroxynitrile lyase from Prunus amygdalus opened a way to large-scale production of this compound. Especially the rationally designed mutation of alanine to glycine at amino acid position 111 of the mature protein tremendously raised the yield for enantioselective conversion of 2-chlorobenzaldehyde to (R)-2-chloromandelonitrile, which can be hydrolysed to the corresponding alpha hydroxy acid. However, expression of this mutein was less efficient than for the unmodified enzyme. Subsequent LC/MS/MS-analysis of the protein sequence revealed that mutation A111G triggered the posttranslational deamidation of the neighbouring residue asparagine (N110) to aspartic acid. This finding on the one hand could explain the decreased secretion efficiency of the mutant as compared to the wildtype enzyme, but on the other hand raised the question which of the two residues was truly accountable for the enhanced conversion. The muteins N110D, A111G and N110DA111G were constructed and compared in terms of protein productivity and performance in chemical syntheses. The expression level of the double mutein was augmented significantly and the enantioselectivity remained high. Reduced protein expression of mutein PaHNL5-L1Q-A111G was remedied by mutational anticipation of posttranslational deamidation.
Subject(s)
Alanine/genetics , Aldehyde-Lyases/metabolism , Genetic Engineering , Glycine/genetics , Protein Processing, Post-Translational , Prunus/enzymology , Aldehyde-Lyases/genetics , Amino Acid Substitution , Benzaldehydes/chemistry , Catalysis , Electrophoresis, Polyacrylamide Gel , Halogens , Kinetics , Mutant Proteins/biosynthesis , Mutant Proteins/metabolism , Nitriles , Subcellular Fractions/enzymologyABSTRACT
N-Glycosylation of proteins is a common posttranslational modification in eukaryotes. Often this results in enhanced protein stability through protection by the attached sugar moieties. Due to its 13 potential N-glycosylation motifs (N-X-T/S), recombinant hydroxynitrile lyase isoenzyme 5 from almonds (PaHNL5) is secreted by the heterologous host Pichia pastoris in a massively glycosylated form, and it shows extraordinary stability at low pH. The importance of N-glycosylation in general, and individual glycosylation sites in particular for stability at low pH were investigated. To identify especially important glycosylation sites asparagine from all N-X-S/T-motifs was replaced by serine. Thus, critical sites, which contributed to overall enzyme activity and/or stability, were identified individually. One glycosylation site revealed to be essential for stability at low pH. After enzymatic deglycosylation, leaving only one acetylglucosamine attached to asparagines, PaHNL5 retained most of its stability at low pH. Protonation effects in the active site as well as higher-order aggregational events upon incubation in low pH were excluded. This study provides evidence for the interconnection of N-glycosylation and stability at low pH for PaHNL5. Moreover, serine scanning was proven to be applicable for quick identification of critical glycosylation sites.
