title: Counteracting expression deficiencies by anticipating posttranslational modification of PaHNL5-L1Q-A111G by genetic engineering.
abstract: (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.
authors.affiliation: Research Centre Applied Biocatalysis, Institute of Organic Chemistry, Graz University of Technology, Stremayrgasse 16, A-8010 Graz, Austria.
authors.forename: Richard
authors.initials: R
authors.lastname: Gaisberger
authors.forename: Roland
authors.initials: R
authors.lastname: Weis
authors.forename: Ruud
authors.initials: R
authors.lastname: Luiten
authors.forename: Wolfgang
authors.initials: W
authors.lastname: Skranc
authors.forename: Marcel
authors.initials: M
authors.lastname: Wubbolts
authors.forename: Herfried
authors.initials: H
authors.lastname: Griengl
authors.forename: Anton
authors.initials: A
authors.lastname: Glieder
mesh_terms: D000409:Alanine / Q000235:genetics*; D000446:Aldehyde-Lyases / Q000235:genetics / Q000378:metabolism*; D019943:Amino Acid Substitution; D001547:Benzaldehydes / Q000737:chemistry; D002384:Catalysis; D004591:Electrophoresis, Polyacrylamide Gel; D005818:Genetic Engineering; D005998:Glycine / Q000235:genetics*; D006219:Halogens; D007700:Kinetics; D050505:Mutant Proteins / Q000096:biosynthesis / Q000378:metabolism; D009570:Nitriles; D011499:Protein Processing, Post-Translational; D027861:Prunus / Q000201:enzymology*; D013347:Subcellular Fractions / Q000201:enzymology
chemical_list: D001547:Benzaldehydes; D006219:Halogens; D050505:Mutant Proteins; D009570:Nitriles; D000446:Aldehyde-Lyases; D000409:Alanine; C032175:benzaldehyde; D005998:Glycine