Introducing transgalactosylation activity into a family 42 β-galactosidase.
Chemo-enzymatic synthesis of oligosaccharides exploits the diversity of glycosidases and their ability to promote transglycosylation reactions in parallel with hydrolysis. Methods to increase the transglycosylation/hydrolysis ratio include site-directed mutagenesis and medium modification. The former approach was successful in several cases and has provided the best synthetic yields with glycosynthases-mutants at the catalytic nucleophile position that promote transglycosylation with high efficiency, but do not hydrolyze the oligosaccharide products. Several glycosidases have proven recalcitrant to this conversion, thus alternative methods to increase the transglycosylation/hydrolysis ratio by mutation would be very useful. Here we show that a mutant of a β-galactosidase from Alicyclobacillus acidocaldarius in an invariant residue in the active site of the enzymes of this family (glutamic acid 361) carries out efficient transglycosylation reactions on different acceptors only in the presence of external ions with yields up to 177-fold higher than that of the wild type. This is the first case in which sodium azide and sodium formate in combination with site-directed mutagenesis have been used to introduce transglycosylation activity into a glycosidase. These observations will hopefully guide further efforts to generate useful synthases.
Institute of Biosciences and Bioresources, National Research Council of Italy, Via P. Castellino 111, 80131 Naples, Italy.|Department of Biology, University of Naples "Federico II", Complesso Universitario di Monte S. Angelo, Via Cupa Nuova Cinthia 21, 80126 Napoli, Italy.
Andrea
A
Strazzulli
Institute of Biosciences and Bioresources, National Research Council of Italy, Via P. Castellino 111, 80131 Naples, Italy.
Beatrice
B
Cobucci-Ponzano
Department of Chemical Sciences, University of Naples "Federico II", Complesso Universitario di Monte S. Angelo, Via Cupa Nuova Cinthia 21, 80126 Napoli, Italy.
Sara
S
Carillo
Department of Chemical Sciences, University of Naples "Federico II", Complesso Universitario di Monte S. Angelo, Via Cupa Nuova Cinthia 21, 80126 Napoli, Italy.
Emiliano
E
Bedini
Department of Chemical Sciences, University of Naples "Federico II", Complesso Universitario di Monte S. Angelo, Via Cupa Nuova Cinthia 21, 80126 Napoli, Italy.
Maria Michela
MM
Corsaro
Institute of Biosciences and Bioresources, National Research Council of Italy, Via P. Castellino 111, 80131 Naples, Italy.
Gabriella
G
Pocsfalvi
Department of Chemistry, University of British Columbia, 2036 Main Mall, Vancouver, British Columbia V6T 1Z1, Canada.
Stephen G
SG
Withers
Institute of Biosciences and Bioresources, National Research Council of Italy, Via P. Castellino 111, 80131 Naples, Italy.
Mosè
M
Rossi
Institute of Biosciences and Bioresources, National Research Council of Italy, Via P. Castellino 111, 80131 Naples, Italy.|Department of Biology, University of Naples "Federico II", Complesso Universitario di Monte S. Angelo, Via Cupa Nuova Cinthia 21, 80126 Napoli, Italy.
Marco
M
Moracci
D056450:Alicyclobacillus / Q000201:enzymology* / Q000235:genetics; D019943:Amino Acid Substitution; D002384:Catalysis; D020134:Catalytic Domain; D006031:Glycosylation; D006868:Hydrolysis; D007700:Kinetics; D009154:Mutation; D009844:Oligosaccharides / Q000096:biosynthesis / Q000737:chemistry*; D013379:Substrate Specificity; D001616:beta-Galactosidase / Q000737:chemistry* / Q000235:genetics
D009844:Oligosaccharides; D001616:beta-Galactosidase
chemical rescue; glycosynthase; oligosaccharide synthesis; thermophilic glycosidases; transgalactosylation