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 (2020)
An α2,3‐sialyltransferase from Photobacterium phosphoreum with broad substrate scope: controlling hydrolytic activity by directed evolution.
In: Chemistry – a European Journal, 26 (50)
doi: 10.1002/chem.202002277
Artikel, Bibliographie

Dies ist die neueste Version dieses Eintrags.

Kurzbeschreibung (Abstract)

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 (2,3SiaTpph) 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 2,3SiaTpph (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.

Typ des Eintrags:

Artikel

Erschienen:

2020

Autor(en):

Mertsch, Alexander ; He, Ning ; Yi, Dong ; Kickstein, Michael ; Fessner, Wolf‐Dieter

Art des Eintrags:

Bibliographie

Titel:

An α2,3‐sialyltransferase from Photobacterium phosphoreum with broad substrate scope: controlling hydrolytic activity by directed evolution

Sprache:

Englisch

Publikationsjahr:

2020

Ort:

Weinheim

Verlag:

Wiley-VCH

Titel der Zeitschrift, Zeitung oder Schriftenreihe:

Chemistry – a European Journal

Jahrgang/Volume einer Zeitschrift:

(Heft-)Nummer:

DOI:

10.1002/chem.202002277

Zugehörige Links:

TUprints Zweitveröffentlichung

Kurzbeschreibung (Abstract):

Alternatives oder übersetztes Abstract:

Alternatives Abstract

Sprache

Be tolerant: An engineered sialyltransferase has exceptional tolerance for a wide variety of substrate modifications, both in glycosyl donor and acceptor moieties. Its undesired promiscuous hydrolytic activity was significantly reduced through deep mutational scanning around the active site. The resultant enzyme is a powerful catalyst for the one-pot cascade synthesis of novel molecular probes for biological studies.

Englisch

Freie Schlagworte:

biocatalysis, carbohydrates, enzyme promiscuity, protein engineering, sialoconjugates

Sachgruppe der Dewey Dezimalklassifikatin (DDC):

500 Naturwissenschaften und Mathematik > 540 Chemie

Fachbereich(e)/-gebiet(e):

07 Fachbereich Chemie
07 Fachbereich Chemie > Clemens-Schöpf-Institut
07 Fachbereich Chemie > Clemens-Schöpf-Institut > Fachgebiet Organische Chemie

Hinterlegungsdatum:

29 Jan 2024 06:52

Letzte Änderung:

01 Feb 2024 07:50

PPN: