Modulating the regioselectivity of a Pasteurella multocida sialyltransferase for biocatalytic production of 3'- and 6'-sialyllactose

Yao Guo, Carsten Jers, Anne S. Meyer, Haiying Li, Finn Kirpekar, Jørn Dalgaard Mikkelsen

Research output: Contribution to journalJournal articleResearchpeer-review

Abstract

Several bacterial sialyltransferases have been reported to be multifunctional also catalysing sialidase and trans-sialidase reactions. In this study, we examined the trans-sialylation efficacy and regioselectivity of mutants of the multifunctional Pasteurella multocida sialyltransferase (PmST) for catalysing the synthesis of 3'- and 6'-sialyllactose using casein glycomacropeptide as sialyl-donor and lactose as acceptor. The mutation P34H led to a 980-fold increase in α-2,6-sialyltransferase activity (with cytidine-5'-monophospho-N-acetylneuraminic acid as donor), while its α-2,3-sialyltransferase activity was abolished. Histidine in this position is conserved in α-2,6-sialyltransferases and has been suggested, and recently confirmed, to be the determinant for strict regiospecificity in the sialyltransferase reaction. Our data verified this theorem. In trans-sialidase reactions, the P34H mutant displayed a distinct preference for 6'-sialyllactose synthesis but low levels of 3'-sialyllactose were also produced. The sialyllactose yield was however lower than when using PmSTWT under optimal conditions for 6'-sialyllactose formation. The discrepancy in regiospecificity between the two reactions could indicate subtle differences in the substrate binding site in the two reactions. In contrast, the two mutations E271F and R313Y led to preferential synthesis of 3'-sialyllactose over 6'-sialyllactose and the double mutant (PmSTE271F/R313Y) exhibited the highest α-2,3-regioselectivity via reduced sialidase and α-2,6-trans-sialidase activity. The double mutant PmSTE271F/R313Y thus showed the highest α-2,3-regioselectivity and constitutes an interesting enzyme for regioselective synthesis of α-2,3-sialylated glycans. This study has expanded the understanding of the structure-function relationship of multifunctional, bacterial sialyltransferases and provided new enzymes for regioselective glycan sialylation.
Original languageEnglish
JournalEnzyme and Microbial Technology
Volume78
Pages (from-to)54-62
ISSN0141-0229
DOIs
Publication statusPublished - 2015

Keywords

  • Pasteurella multocida
  • Regioselectivity
  • Sialyllactose
  • Sialyltransferase
  • Site-directed mutagenesis
  • Trans-sialidase

Cite this

@article{f750422989b94900811361578759de8a,
title = "Modulating the regioselectivity of a Pasteurella multocida sialyltransferase for biocatalytic production of 3'- and 6'-sialyllactose",
abstract = "Several bacterial sialyltransferases have been reported to be multifunctional also catalysing sialidase and trans-sialidase reactions. In this study, we examined the trans-sialylation efficacy and regioselectivity of mutants of the multifunctional Pasteurella multocida sialyltransferase (PmST) for catalysing the synthesis of 3'- and 6'-sialyllactose using casein glycomacropeptide as sialyl-donor and lactose as acceptor. The mutation P34H led to a 980-fold increase in α-2,6-sialyltransferase activity (with cytidine-5'-monophospho-N-acetylneuraminic acid as donor), while its α-2,3-sialyltransferase activity was abolished. Histidine in this position is conserved in α-2,6-sialyltransferases and has been suggested, and recently confirmed, to be the determinant for strict regiospecificity in the sialyltransferase reaction. Our data verified this theorem. In trans-sialidase reactions, the P34H mutant displayed a distinct preference for 6'-sialyllactose synthesis but low levels of 3'-sialyllactose were also produced. The sialyllactose yield was however lower than when using PmSTWT under optimal conditions for 6'-sialyllactose formation. The discrepancy in regiospecificity between the two reactions could indicate subtle differences in the substrate binding site in the two reactions. In contrast, the two mutations E271F and R313Y led to preferential synthesis of 3'-sialyllactose over 6'-sialyllactose and the double mutant (PmSTE271F/R313Y) exhibited the highest α-2,3-regioselectivity via reduced sialidase and α-2,6-trans-sialidase activity. The double mutant PmSTE271F/R313Y thus showed the highest α-2,3-regioselectivity and constitutes an interesting enzyme for regioselective synthesis of α-2,3-sialylated glycans. This study has expanded the understanding of the structure-function relationship of multifunctional, bacterial sialyltransferases and provided new enzymes for regioselective glycan sialylation.",
keywords = "Pasteurella multocida, Regioselectivity, Sialyllactose, Sialyltransferase, Site-directed mutagenesis, Trans-sialidase",
author = "Yao Guo and Carsten Jers and Meyer, {Anne S.} and Haiying Li and Finn Kirpekar and Mikkelsen, {J{\o}rn Dalgaard}",
year = "2015",
doi = "10.1016/j.enzmictec.2015.06.012",
language = "English",
volume = "78",
pages = "54--62",
journal = "Enzyme and Microbial Technology",
issn = "0141-0229",
publisher = "Elsevier",

}

Modulating the regioselectivity of a Pasteurella multocida sialyltransferase for biocatalytic production of 3'- and 6'-sialyllactose. / Guo, Yao; Jers, Carsten; Meyer, Anne S.; Li, Haiying; Kirpekar, Finn; Mikkelsen, Jørn Dalgaard.

In: Enzyme and Microbial Technology, Vol. 78, 2015, p. 54-62.

Research output: Contribution to journalJournal articleResearchpeer-review

TY - JOUR

T1 - Modulating the regioselectivity of a Pasteurella multocida sialyltransferase for biocatalytic production of 3'- and 6'-sialyllactose

AU - Guo, Yao

AU - Jers, Carsten

AU - Meyer, Anne S.

AU - Li, Haiying

AU - Kirpekar, Finn

AU - Mikkelsen, Jørn Dalgaard

PY - 2015

Y1 - 2015

N2 - Several bacterial sialyltransferases have been reported to be multifunctional also catalysing sialidase and trans-sialidase reactions. In this study, we examined the trans-sialylation efficacy and regioselectivity of mutants of the multifunctional Pasteurella multocida sialyltransferase (PmST) for catalysing the synthesis of 3'- and 6'-sialyllactose using casein glycomacropeptide as sialyl-donor and lactose as acceptor. The mutation P34H led to a 980-fold increase in α-2,6-sialyltransferase activity (with cytidine-5'-monophospho-N-acetylneuraminic acid as donor), while its α-2,3-sialyltransferase activity was abolished. Histidine in this position is conserved in α-2,6-sialyltransferases and has been suggested, and recently confirmed, to be the determinant for strict regiospecificity in the sialyltransferase reaction. Our data verified this theorem. In trans-sialidase reactions, the P34H mutant displayed a distinct preference for 6'-sialyllactose synthesis but low levels of 3'-sialyllactose were also produced. The sialyllactose yield was however lower than when using PmSTWT under optimal conditions for 6'-sialyllactose formation. The discrepancy in regiospecificity between the two reactions could indicate subtle differences in the substrate binding site in the two reactions. In contrast, the two mutations E271F and R313Y led to preferential synthesis of 3'-sialyllactose over 6'-sialyllactose and the double mutant (PmSTE271F/R313Y) exhibited the highest α-2,3-regioselectivity via reduced sialidase and α-2,6-trans-sialidase activity. The double mutant PmSTE271F/R313Y thus showed the highest α-2,3-regioselectivity and constitutes an interesting enzyme for regioselective synthesis of α-2,3-sialylated glycans. This study has expanded the understanding of the structure-function relationship of multifunctional, bacterial sialyltransferases and provided new enzymes for regioselective glycan sialylation.

AB - Several bacterial sialyltransferases have been reported to be multifunctional also catalysing sialidase and trans-sialidase reactions. In this study, we examined the trans-sialylation efficacy and regioselectivity of mutants of the multifunctional Pasteurella multocida sialyltransferase (PmST) for catalysing the synthesis of 3'- and 6'-sialyllactose using casein glycomacropeptide as sialyl-donor and lactose as acceptor. The mutation P34H led to a 980-fold increase in α-2,6-sialyltransferase activity (with cytidine-5'-monophospho-N-acetylneuraminic acid as donor), while its α-2,3-sialyltransferase activity was abolished. Histidine in this position is conserved in α-2,6-sialyltransferases and has been suggested, and recently confirmed, to be the determinant for strict regiospecificity in the sialyltransferase reaction. Our data verified this theorem. In trans-sialidase reactions, the P34H mutant displayed a distinct preference for 6'-sialyllactose synthesis but low levels of 3'-sialyllactose were also produced. The sialyllactose yield was however lower than when using PmSTWT under optimal conditions for 6'-sialyllactose formation. The discrepancy in regiospecificity between the two reactions could indicate subtle differences in the substrate binding site in the two reactions. In contrast, the two mutations E271F and R313Y led to preferential synthesis of 3'-sialyllactose over 6'-sialyllactose and the double mutant (PmSTE271F/R313Y) exhibited the highest α-2,3-regioselectivity via reduced sialidase and α-2,6-trans-sialidase activity. The double mutant PmSTE271F/R313Y thus showed the highest α-2,3-regioselectivity and constitutes an interesting enzyme for regioselective synthesis of α-2,3-sialylated glycans. This study has expanded the understanding of the structure-function relationship of multifunctional, bacterial sialyltransferases and provided new enzymes for regioselective glycan sialylation.

KW - Pasteurella multocida

KW - Regioselectivity

KW - Sialyllactose

KW - Sialyltransferase

KW - Site-directed mutagenesis

KW - Trans-sialidase

U2 - 10.1016/j.enzmictec.2015.06.012

DO - 10.1016/j.enzmictec.2015.06.012

M3 - Journal article

C2 - 26215345

VL - 78

SP - 54

EP - 62

JO - Enzyme and Microbial Technology

JF - Enzyme and Microbial Technology

SN - 0141-0229

ER -