Loop Protein Engineering for Improved Transglycosylation Activity of a β‐N‐Acetylhexosaminidase

• Published in: Chembiochem : a European journal of chemical biology 2018-09, Vol.19 (17), p.1858-1865
• Main Authors: Jamek, Shariza B., Muschiol, Jan, Holck, Jesper, Zeuner, Birgitte, Busk, Peter K., Mikkelsen, Jørn D., Meyer, Anne S.
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Bacteria - enzymology; Bacterial Proteins - chemistry; Bacterial Proteins - genetics; beta-N-Acetylhexosaminidases - chemistry; beta-N-Acetylhexosaminidases - genetics; Breast milk; Catalytic Domain; Disaccharides - chemistry; Enzymes; Escherichia coli - genetics; glucosamines; Glycoside hydrolase; Glycosylation; Glycosyltransferases - chemistry; Glycosyltransferases - genetics; Homology; Hydrolase; hydrolases; Hydrolysis; Insertion; Lactose; Lactose - chemistry; Mutants; N-Acetylglucosamine; Nucleotide sequence; Oligosaccharides; Pattern analysis; Pattern recognition; peptide pattern recognition; Protein Conformation; Protein engineering; Protein Engineering - methods; Proteins; Sequence Alignment; Sequences; Shielding; Subgroups; Substrates; transglycosylation; Trisaccharides - chemical synthesis
• ISSN: 1439-4227; 1439-7633
• DOI: 10.1002/cbic.201800181

Certain enzymes of the glycoside hydrolase family 20 (GH20) exert transglycosylation activity and catalyze the transfer of β‐N‐acetylglucosamine (GlcNAc) from a chitobiose donor to lactose to produce lacto‐N‐triose II (LNT2), a key human milk oligosaccharide backbone moiety. The present work is aimed at increasing the transglycosylation activity of two selected hexosaminidases, HEX1 and HEX2, to synthesize LNT2 from lactose and chitobiose. Peptide pattern recognition analysis was used to categorize all GH20 proteins in subgroups. On this basis, we identified a series of proteins related to HEX1 and HEX2. By sequence alignment, four additional loop sequences were identified that were not present in HEX1 and HEX2. Insertion of these loop sequences into the wild‐type sequences induced increased transglycosylation activity for three out of eight mutants. The best mutant, HEX1GTEPG, had a transglycosylation yield of LNT2 on the donor that was nine times higher than that of the wild‐type enzyme. Homology modeling of the enzymes revealed that the loop insertion produced a more shielded substrate‐binding pocket. This shielding is suggested to explain the reduced hydrolytic activity, which in turn resulted in the increased transglycosylation activity of HEX1GTEPG. Transglycosylation by loop protein engineering: A β‐N‐acetylhexosaminidase is engineered towards increased transglycosylation activity by introduction of a short five amino‐acid loop close to the active site. The best variant gives a yield of lacto‐N‐triose II that is nine times higher than that of the wild‐type enzyme.