Inter domain linker region affects properties of CBM6 in GH5_34 arabinoxylanases and alters oligosaccharide product profile
Norlander, Siri LU ; Jasilionis, Andrius LU ; Allahgholi, Leila LU ; Wennerberg, Christina LU ; Grey, Carl LU ; Adlercreutz, Patrick LU
and Karlsson, Eva Nordberg
LU
(2024)
In Glycobiology
34(8).
- Abstract
Understanding the relation between enzyme domain structure and catalytic activity is crucial for optimal engineering of novel enzymes for lignocellulose bioconversion. Xylanases with varying specificities are commonly used to valorise the hemicellulose arabinoxylan (AX), yet characterization of specific arabinoxylanases remain limited. Two homologous GH5_34 arabinoxylanases, HhXyn5A and CtXyn5A, in which the two domains are connected by a 40-residue linker, exhibit distinct activity on AX, yielding different reaction product patterns, despite high sequence identity, conserved active sites and similar domain composition. In this study, the carbohydrate binding module 6 (CBM6), or the inter domain linker together with CBM6, were swapped... (More)
Understanding the relation between enzyme domain structure and catalytic activity is crucial for optimal engineering of novel enzymes for lignocellulose bioconversion. Xylanases with varying specificities are commonly used to valorise the hemicellulose arabinoxylan (AX), yet characterization of specific arabinoxylanases remain limited. Two homologous GH5_34 arabinoxylanases, HhXyn5A and CtXyn5A, in which the two domains are connected by a 40-residue linker, exhibit distinct activity on AX, yielding different reaction product patterns, despite high sequence identity, conserved active sites and similar domain composition. In this study, the carbohydrate binding module 6 (CBM6), or the inter domain linker together with CBM6, were swapped to investigate their influence on hydrolytic activity and oligosaccharide product pattern on cereal AXs. The variants, with only CBM6 swapped, displayed reduced activity on commercial wheat and rye AX, as well as on extracted oat fibre, compared to the original enzymes. Additionally, exchange of both linker and CBM6 resulted in a reduced ratio of enzyme produced in soluble form in Escherichia coli cultivations, causing loss of activity of both HhXyn5A and CtXyn5A variants. Analysis of oligosaccharide product patterns applying HPAEC–PAD revealed a decreased number of reaction products for CtXyn5A with swapped CBM6, which resembled the product pattern of HhXyn5A. These findings emphasize the importance of the CBM6 interactions with the linker and the catalytic domain for enzyme activity and specificity, and underlines the role of the linker in enzyme structure organisation and product formation, where alterations in linker interactions with the catalytic and/or CBM6 domains, influence enzyme-substrate association and specificity.
(Less)
- author
- Norlander, Siri
LU
; Jasilionis, Andrius
LU
; Allahgholi, Leila
LU
; Wennerberg, Christina
LU
; Grey, Carl
LU
; Adlercreutz, Patrick
LU
and Karlsson, Eva Nordberg
LU
- organization
- publishing date
- 2024-08
- type
- Contribution to journal
- publication status
- published
- subject
- keywords
- affinity gel electrophoresis, arabinoxylanase, carbohydrate binding module, domain swapping
- in
- Glycobiology
- volume
- 34
- issue
- 8
- article number
- cwae048
- publisher
- Oxford University Press
- external identifiers
-
- pmid:38982733
- scopus:85198736665
- ISSN
- 0959-6658
- DOI
- 10.1093/glycob/cwae048
- language
- English
- LU publication?
- yes
- id
- 1e75a15a-84b7-495e-b89c-8ef436c3df9a
- date added to LUP
- 2024-09-09 15:38:46
- date last changed
- 2024-09-10 03:04:13
@article{1e75a15a-84b7-495e-b89c-8ef436c3df9a,
abstract = {{<p>Understanding the relation between enzyme domain structure and catalytic activity is crucial for optimal engineering of novel enzymes for lignocellulose bioconversion. Xylanases with varying specificities are commonly used to valorise the hemicellulose arabinoxylan (AX), yet characterization of specific arabinoxylanases remain limited. Two homologous GH5_34 arabinoxylanases, HhXyn5A and CtXyn5A, in which the two domains are connected by a 40-residue linker, exhibit distinct activity on AX, yielding different reaction product patterns, despite high sequence identity, conserved active sites and similar domain composition. In this study, the carbohydrate binding module 6 (CBM6), or the inter domain linker together with CBM6, were swapped to investigate their influence on hydrolytic activity and oligosaccharide product pattern on cereal AXs. The variants, with only CBM6 swapped, displayed reduced activity on commercial wheat and rye AX, as well as on extracted oat fibre, compared to the original enzymes. Additionally, exchange of both linker and CBM6 resulted in a reduced ratio of enzyme produced in soluble form in Escherichia coli cultivations, causing loss of activity of both HhXyn5A and CtXyn5A variants. Analysis of oligosaccharide product patterns applying HPAEC–PAD revealed a decreased number of reaction products for CtXyn5A with swapped CBM6, which resembled the product pattern of HhXyn5A. These findings emphasize the importance of the CBM6 interactions with the linker and the catalytic domain for enzyme activity and specificity, and underlines the role of the linker in enzyme structure organisation and product formation, where alterations in linker interactions with the catalytic and/or CBM6 domains, influence enzyme-substrate association and specificity.</p>}},
author = {{Norlander, Siri and Jasilionis, Andrius and Allahgholi, Leila and Wennerberg, Christina and Grey, Carl and Adlercreutz, Patrick and Karlsson, Eva Nordberg}},
issn = {{0959-6658}},
keywords = {{affinity gel electrophoresis; arabinoxylanase; carbohydrate binding module; domain swapping}},
language = {{eng}},
number = {{8}},
publisher = {{Oxford University Press}},
series = {{Glycobiology}},
title = {{Inter domain linker region affects properties of CBM6 in GH5_34 arabinoxylanases and alters oligosaccharide product profile}},
url = {{http://dx.doi.org/10.1093/glycob/cwae048}},
doi = {{10.1093/glycob/cwae048}},
volume = {{34}},
year = {{2024}},
}