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

Shariza B. Jamek, Jan Muschiol, Jesper Holck, Birgitte Zeuner, Peter K. Busk, Jørn D. Mikkelsen, Anne S. Meyer*

*Corresponding author for this work

Research output: Contribution to journalJournal articleResearchpeer-review

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, 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.
Original languageEnglish
JournalChembiochem
Volume19
Issue number17
Pages (from-to)1858-1865
Number of pages9
ISSN1439-4227
DOIs
Publication statusPublished - 2018

Keywords

  • Glucosamines
  • Hydrolases
  • Oligosaccharides
  • Peptide pattern recognition
  • Transglycosylation

Cite this

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title = "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, 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.",
keywords = "Glucosamines, Hydrolases, Oligosaccharides, Peptide pattern recognition, Transglycosylation",
author = "Jamek, {Shariza B.} and Jan Muschiol and Jesper Holck and Birgitte Zeuner and Busk, {Peter K.} and Mikkelsen, {J{\o}rn D.} and Meyer, {Anne S.}",
year = "2018",
doi = "10.1002/cbic.201800181",
language = "English",
volume = "19",
pages = "1858--1865",
journal = "ChemBioChem",
issn = "1439-4227",
publisher = "Wiley - V C H Verlag GmbH & Co. KGaA",
number = "17",

}

Loop Protein Engineering for Improved Transglycosylation Activity of a β-N -Acetylhexosaminidase. / Jamek, Shariza B.; Muschiol, Jan; Holck, Jesper; Zeuner, Birgitte; Busk, Peter K.; Mikkelsen, Jørn D.; Meyer, Anne S.

In: Chembiochem, Vol. 19, No. 17, 2018, p. 1858-1865.

Research output: Contribution to journalJournal articleResearchpeer-review

TY - JOUR

T1 - Loop Protein Engineering for Improved Transglycosylation Activity of a β-N -Acetylhexosaminidase

AU - Jamek, Shariza B.

AU - Muschiol, Jan

AU - Holck, Jesper

AU - Zeuner, Birgitte

AU - Busk, Peter K.

AU - Mikkelsen, Jørn D.

AU - Meyer, Anne S.

PY - 2018

Y1 - 2018

N2 - 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.

AB - 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.

KW - Glucosamines

KW - Hydrolases

KW - Oligosaccharides

KW - Peptide pattern recognition

KW - Transglycosylation

U2 - 10.1002/cbic.201800181

DO - 10.1002/cbic.201800181

M3 - Journal article

C2 - 29911342

VL - 19

SP - 1858

EP - 1865

JO - ChemBioChem

JF - ChemBioChem

SN - 1439-4227

IS - 17

ER -