Structural insights of RmXyn10A - A prebiotic-producing GH10 xylanase with a non-conserved aglycone binding region

Anna Aronsson, Fatma Guler, Maxim Petoukhov, Susan Crennell, Dmitri i. Svergun, javier Linares-Pasten, Eva Nordbery Karlsson

Research output: Contribution to journalArticle

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Abstract

Hydrolysis of arabinoxylan (AX) by glycoside hydrolase family 10 (GH10) xylanases produces xylo- and arabinoxylo-oligosaccharides ((A)XOS) which have shown prebiotic effects. The thermostable GH10 xylanase RmXyn10A has shown great potential to produce (A)XOS. In this study, the structure of RmXyn10A was investigated, the catalytic module by homology modelling and site-directed mutagenesis and the arrangement of its five domains by small-angle X-ray scattering (SAXS). Substrate specificity was explored in silico by manual docking and molecular dynamic simulations. It has been shown in the literature that the glycone subsites of GH10 xylanases are well conserved and our results suggest that RmXyn10A is no exception. The aglycone subsites are less investigated, and the modelled structure of RmXyn10A suggests that loop β6α6 in the aglycone part of the active site contains a non-conserved α-helix, which blocks the otherwise conserved space of subsite +2. This structural feature has only been observed for one other GH10 xylanase. In RmXyn10A, docking revealed two alternative binding regions, one on either side of the α-helix. However, only one was able to accommodate arabinose-substitutions and the mutation study suggests that the same region is responsible for binding XOS. Several non-conserved structural features are most likely to be responsible for providing affinity for arabinose-substitutions in subsites +1 and +2. The SAXS rigid model of the modular arrangement of RmXyn10A displays the catalytic module close to the cell-anchoring domain while the carbohydrate binding modules are further away, likely explaining the observed lack of contribution of the CBMs to activity.
Original languageEnglish
Pages (from-to)292-306
JournalBiochimica Et Biophysica Acta-Proteins and Proteomics
Volume1866
Issue number2
Early online date14 Nov 2017
DOIs
Publication statusPublished - Feb 2018

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Prebiotics
Glycoside Hydrolases
Arabinose
X ray scattering
Substitution reactions
X-Rays
Mutagenesis
Molecular Dynamics Simulation
Substrate Specificity
Site-Directed Mutagenesis
Oligosaccharides
Computer Simulation
Molecular dynamics
Hydrolysis
Catalytic Domain
Display devices
Carbohydrates
Mutation
Computer simulation
Substrates

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Structural insights of RmXyn10A - A prebiotic-producing GH10 xylanase with a non-conserved aglycone binding region. / Aronsson, Anna; Guler, Fatma ; Petoukhov, Maxim; Crennell, Susan; Svergun, Dmitri i.; Linares-Pasten, javier; Nordbery Karlsson, Eva.

In: Biochimica Et Biophysica Acta-Proteins and Proteomics, Vol. 1866, No. 2, 02.2018, p. 292-306.

Research output: Contribution to journalArticle

Aronsson, Anna ; Guler, Fatma ; Petoukhov, Maxim ; Crennell, Susan ; Svergun, Dmitri i. ; Linares-Pasten, javier ; Nordbery Karlsson, Eva. / Structural insights of RmXyn10A - A prebiotic-producing GH10 xylanase with a non-conserved aglycone binding region. In: Biochimica Et Biophysica Acta-Proteins and Proteomics. 2018 ; Vol. 1866, No. 2. pp. 292-306.
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AU - Aronsson, Anna

AU - Guler, Fatma

AU - Petoukhov, Maxim

AU - Crennell, Susan

AU - Svergun, Dmitri i.

AU - Linares-Pasten, javier

AU - Nordbery Karlsson, Eva

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AB - Hydrolysis of arabinoxylan (AX) by glycoside hydrolase family 10 (GH10) xylanases produces xylo- and arabinoxylo-oligosaccharides ((A)XOS) which have shown prebiotic effects. The thermostable GH10 xylanase RmXyn10A has shown great potential to produce (A)XOS. In this study, the structure of RmXyn10A was investigated, the catalytic module by homology modelling and site-directed mutagenesis and the arrangement of its five domains by small-angle X-ray scattering (SAXS). Substrate specificity was explored in silico by manual docking and molecular dynamic simulations. It has been shown in the literature that the glycone subsites of GH10 xylanases are well conserved and our results suggest that RmXyn10A is no exception. The aglycone subsites are less investigated, and the modelled structure of RmXyn10A suggests that loop β6α6 in the aglycone part of the active site contains a non-conserved α-helix, which blocks the otherwise conserved space of subsite +2. This structural feature has only been observed for one other GH10 xylanase. In RmXyn10A, docking revealed two alternative binding regions, one on either side of the α-helix. However, only one was able to accommodate arabinose-substitutions and the mutation study suggests that the same region is responsible for binding XOS. Several non-conserved structural features are most likely to be responsible for providing affinity for arabinose-substitutions in subsites +1 and +2. The SAXS rigid model of the modular arrangement of RmXyn10A displays the catalytic module close to the cell-anchoring domain while the carbohydrate binding modules are further away, likely explaining the observed lack of contribution of the CBMs to activity.

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SN - 1570-9639

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