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

Abstract

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, HEX1 , 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 HEX1 .

β-N-Acetylhexosaminidases for Carbohydrate Synthesis via Trans-Glycosylation

“…Lastly, we would like to highlight a potential hotspot for mutations that drive trans-glycosylation activity in GH20 enzymes. We previously demonstrated that introduction of a specific loop, which was identified in related GH20 sequences from pathogens, into Hex1 led to a >5-fold increased trans-glycosylation product yield in reactions with Lac as acceptor and (GlcNAc) 2 as donor ( Figure 7B, Table 8) [117]. Additionally, in a recent mutational study on BbhI, the mutant W805R ( Figure 7A) was identified as the one with superior trans-glycosylation activity [104].…”

“…Although chitin, e.g., from shrimp or crab waste streams, may seem an obvious substrate for sustainable β-N-acetylhexosaminidase catalyzed trans- Figure 7. Active sites models of two bacterial β-N-acetylhexosaminidases: (A) homology model of BbhI from Bifidobacterium bifidum (created with YASARA [138]) with the mutated W805 [104] highlighted in orange and GlcNAc bound in the active site; (B) homology model of Hex1GTEPG [117] with the inserted loop highlighted in red and the newly positioned R360 in orange; the reaction intermediate Glc-oxa was docked into the structure [117]. The catalytic Asp-Glu pair is highlighted in yellow, the water-stabilizing Tyr residue is highlighted in purple, bound or docked ligands are highlighted in green, and conserved residues (Arg and Trp) are highlighted in dark blue.…”

“…Only the additional loops carrying a negative charge in the middle of the five amino acid sequence (GTEPG and GTDDA) led to an increased trans-glycosylation yield. Other tested loops carrying a positive charge (SFRTP) or a negative charge in a different position (DFVTP) led to a decreased trans-glycosylation activity [117].…”

“…However, in case of Hex1 the inserted loop sequence itself also seems to have an important role with respect to trans-glycosylation activity, since not all alternative loop sequences of the same length were as beneficial as others [117]. Only the additional loops carrying a negative charge in the middle of the five amino acid sequence (GTEPG and GTDDA) led to an increased trans-glycosylation yield.…”

Comparative Characterization of Aspergillus Pectin Lyases by Discriminative Substrate Degradation Profiling

“…The different retention times of the compounds are assumed to derive from different positions of the methyl and/or acetyl substitutions. The porous graphite column in use has previously shown the capability to separate different isomers of compounds with identical molecular composition, but with different linkages and/or planarity (Jamek et al, 2018;Mosbech et al, 2018;Zeuner et al, 2018).…”