Cargando…

An α2,3‐Sialyltransferase from Photobacterium phosphoreum with Broad Substrate Scope: Controlling Hydrolytic Activity by Directed Evolution

Defined sialoglycoconjugates are important molecular probes for studying the role of sialylated glycans in biological systems. We show that the α2,3‐sialyltransferase from Photobacterium phosphoreum JT‐ISH‐467 (2,3SiaT(pph)) tolerates a very broad substrate scope for modifications in the sialic acid...

Descripción completa

Detalles Bibliográficos
Autores principales: Mertsch, Alexander, He, Ning, Yi, Dong, Kickstein, Michael, Fessner, Wolf‐Dieter
Formato: Online Artículo Texto
Lenguaje:English
Publicado: John Wiley and Sons Inc. 2020
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7540698/
https://www.ncbi.nlm.nih.gov/pubmed/32596832
http://dx.doi.org/10.1002/chem.202002277
_version_ 1783591259293614080
author Mertsch, Alexander
He, Ning
Yi, Dong
Kickstein, Michael
Fessner, Wolf‐Dieter
author_facet Mertsch, Alexander
He, Ning
Yi, Dong
Kickstein, Michael
Fessner, Wolf‐Dieter
author_sort Mertsch, Alexander
collection PubMed
description Defined sialoglycoconjugates are important molecular probes for studying the role of sialylated glycans in biological systems. We show that the α2,3‐sialyltransferase from Photobacterium phosphoreum JT‐ISH‐467 (2,3SiaT(pph)) tolerates a very broad substrate scope for modifications in the sialic acid part, including bulky amide variation, C5/C9 substitution, and C5 stereoinversion. To reduce the enzyme's hydrolytic activity, which erodes the product yield, an extensive structure‐guided mutagenesis study identified three variants that show up to five times higher catalytic efficiency for sialyltransfer, up to ten times lower efficiency for substrate hydrolysis, and drastically reduced product hydrolysis. Variant 2,3SiaT(pph) (A151D) displayed the best performance overall in the synthesis of the GM3 trisaccharide (α2,3‐Neu5Ac‐Lac) from lactose in a one‐pot, two‐enzyme cascade. Our study demonstrates that several complementary solutions can be found to suppress the common problem of undesired hydrolysis activity of microbial GT80 sialyltransferases. The new enzymes are powerful catalysts for the synthesis of a wide variety of complex natural and new‐to‐nature sialoconjugates for biological studies.
format Online
Article
Text
id pubmed-7540698
institution National Center for Biotechnology Information
language English
publishDate 2020
publisher John Wiley and Sons Inc.
record_format MEDLINE/PubMed
spelling pubmed-75406982020-10-15 An α2,3‐Sialyltransferase from Photobacterium phosphoreum with Broad Substrate Scope: Controlling Hydrolytic Activity by Directed Evolution Mertsch, Alexander He, Ning Yi, Dong Kickstein, Michael Fessner, Wolf‐Dieter Chemistry Full Papers Defined sialoglycoconjugates are important molecular probes for studying the role of sialylated glycans in biological systems. We show that the α2,3‐sialyltransferase from Photobacterium phosphoreum JT‐ISH‐467 (2,3SiaT(pph)) tolerates a very broad substrate scope for modifications in the sialic acid part, including bulky amide variation, C5/C9 substitution, and C5 stereoinversion. To reduce the enzyme's hydrolytic activity, which erodes the product yield, an extensive structure‐guided mutagenesis study identified three variants that show up to five times higher catalytic efficiency for sialyltransfer, up to ten times lower efficiency for substrate hydrolysis, and drastically reduced product hydrolysis. Variant 2,3SiaT(pph) (A151D) displayed the best performance overall in the synthesis of the GM3 trisaccharide (α2,3‐Neu5Ac‐Lac) from lactose in a one‐pot, two‐enzyme cascade. Our study demonstrates that several complementary solutions can be found to suppress the common problem of undesired hydrolysis activity of microbial GT80 sialyltransferases. The new enzymes are powerful catalysts for the synthesis of a wide variety of complex natural and new‐to‐nature sialoconjugates for biological studies. John Wiley and Sons Inc. 2020-08-07 2020-09-04 /pmc/articles/PMC7540698/ /pubmed/32596832 http://dx.doi.org/10.1002/chem.202002277 Text en © 2020 The Authors. Published by Wiley-VCH GmbH This is an open access article under the terms of the http://creativecommons.org/licenses/by-nc/4.0/ License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited and is not used for commercial purposes.
spellingShingle Full Papers
Mertsch, Alexander
He, Ning
Yi, Dong
Kickstein, Michael
Fessner, Wolf‐Dieter
An α2,3‐Sialyltransferase from Photobacterium phosphoreum with Broad Substrate Scope: Controlling Hydrolytic Activity by Directed Evolution
title An α2,3‐Sialyltransferase from Photobacterium phosphoreum with Broad Substrate Scope: Controlling Hydrolytic Activity by Directed Evolution
title_full An α2,3‐Sialyltransferase from Photobacterium phosphoreum with Broad Substrate Scope: Controlling Hydrolytic Activity by Directed Evolution
title_fullStr An α2,3‐Sialyltransferase from Photobacterium phosphoreum with Broad Substrate Scope: Controlling Hydrolytic Activity by Directed Evolution
title_full_unstemmed An α2,3‐Sialyltransferase from Photobacterium phosphoreum with Broad Substrate Scope: Controlling Hydrolytic Activity by Directed Evolution
title_short An α2,3‐Sialyltransferase from Photobacterium phosphoreum with Broad Substrate Scope: Controlling Hydrolytic Activity by Directed Evolution
title_sort α2,3‐sialyltransferase from photobacterium phosphoreum with broad substrate scope: controlling hydrolytic activity by directed evolution
topic Full Papers
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7540698/
https://www.ncbi.nlm.nih.gov/pubmed/32596832
http://dx.doi.org/10.1002/chem.202002277
work_keys_str_mv AT mertschalexander ana23sialyltransferasefromphotobacteriumphosphoreumwithbroadsubstratescopecontrollinghydrolyticactivitybydirectedevolution
AT hening ana23sialyltransferasefromphotobacteriumphosphoreumwithbroadsubstratescopecontrollinghydrolyticactivitybydirectedevolution
AT yidong ana23sialyltransferasefromphotobacteriumphosphoreumwithbroadsubstratescopecontrollinghydrolyticactivitybydirectedevolution
AT kicksteinmichael ana23sialyltransferasefromphotobacteriumphosphoreumwithbroadsubstratescopecontrollinghydrolyticactivitybydirectedevolution
AT fessnerwolfdieter ana23sialyltransferasefromphotobacteriumphosphoreumwithbroadsubstratescopecontrollinghydrolyticactivitybydirectedevolution
AT mertschalexander a23sialyltransferasefromphotobacteriumphosphoreumwithbroadsubstratescopecontrollinghydrolyticactivitybydirectedevolution
AT hening a23sialyltransferasefromphotobacteriumphosphoreumwithbroadsubstratescopecontrollinghydrolyticactivitybydirectedevolution
AT yidong a23sialyltransferasefromphotobacteriumphosphoreumwithbroadsubstratescopecontrollinghydrolyticactivitybydirectedevolution
AT kicksteinmichael a23sialyltransferasefromphotobacteriumphosphoreumwithbroadsubstratescopecontrollinghydrolyticactivitybydirectedevolution
AT fessnerwolfdieter a23sialyltransferasefromphotobacteriumphosphoreumwithbroadsubstratescopecontrollinghydrolyticactivitybydirectedevolution