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A dual catalytic architecture promotes C-2 stereoinversion of CDP–glucose by the CDP–tyvelose 2-epimerase from Thermodesulfatator atlanticus.

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Title: A dual catalytic architecture promotes C-2 stereoinversion of CDP–glucose by the CDP–tyvelose 2-epimerase from Thermodesulfatator atlanticus.
Authors: Rapp, Christian1, van Overtveldt, Stevie2, Sánchez-Murcia, Pedro A.3, Pfeiffer, Martin1, Beerens, Koen2, Merkaš, Magdalena4, Pavkov-Keller, Tea5, Desmet, Tom2,6, Nidetzky, Bernd1,6 bernd.nidetzky@tugraz.at
Source: Journal of Biological Chemistry. May2026, Vol. 302 Issue 5, p1-17. 17p.
Subjects: Epimerases, Epimerization, Molecular dynamics, Enzyme specificity, Crystal structure, Pyrimidine nucleotides, Catalysis
Abstract: The CDP–tyvelose 2-epimerase from Thermodesulfatator atlanticus (TaTyvE) catalyzes the C-2 epimerization of CDP–glucose to CDP–mannose. The enzyme uses NADdependent oxidation–reduction to achieve C-2 configurational inversion of the substrate. Here, we report the 2.60-Å crystal structure of tetrameric TaTyvE with NAD+ bound in all subunits and CDP bound in one (Protein Data Bank code: 9RL0). Binding of CDP orders the Gly197–Trp207 loop, closing over the sugar binding pocket. The active site of the ternary complex is well preorganized, with only moderate induced-fit conformational changes. Molecular dynamics simulations and site-directed mutagenesis suggest that TaTyvE employs a dual catalytic architecture to control substrate specificity. Asn125, within the TNK segment (Thr124–Asn125–Lys126), promotes sampling of catalytically plausible glucose conformations. The VAM segment (Val83–Ala84–Met85) permits broader conformational flexibility for mannose through backbone contacts. This is supported by a modest ~ 12-fold activity reduction in the Q205A variant. In contrast, N125A abolishes activity. Substrate analogs featuring deoxygenation, stereoinversion, or fluorination at the C-4 were synthesized to examine the role of the sugar C4–OH. Simulations indicated that the C4–OH interacts with Val83, Asn125, and Gln205. Activity was lost with CDP–4-deoxy-glucose and minimally recovered with CDP–4-fluoro-glucose. Removing the C5- hydroxymethyl group to restrict substrate positioning flexibility enhanced the reaction rate. Overall, these results highlight the important interplay of structural preorganization and conformational flexibility in TaTyvE for enzyme activity and specificity in the C-2 epimerization of CDP–glucose. [ABSTRACT FROM AUTHOR]
Copyright of Journal of Biological Chemistry is the property of Elsevier B.V. and its content may not be copied or emailed to multiple sites without the copyright holder's express written permission. Additionally, content may not be used with any artificial intelligence tools or machine learning technologies. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)
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DbLabel: Engineering Source
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  Data: A dual catalytic architecture promotes C-2 stereoinversion of CDP–glucose by the CDP–tyvelose 2-epimerase from Thermodesulfatator atlanticus.
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  Data: <searchLink fieldCode="AR" term="%22Rapp%2C+Christian%22">Rapp, Christian</searchLink><relatesTo>1</relatesTo><br /><searchLink fieldCode="AR" term="%22van+Overtveldt%2C+Stevie%22">van Overtveldt, Stevie</searchLink><relatesTo>2</relatesTo><br /><searchLink fieldCode="AR" term="%22Sánchez-Murcia%2C+Pedro+A%2E%22">Sánchez-Murcia, Pedro A.</searchLink><relatesTo>3</relatesTo><br /><searchLink fieldCode="AR" term="%22Pfeiffer%2C+Martin%22">Pfeiffer, Martin</searchLink><relatesTo>1</relatesTo><br /><searchLink fieldCode="AR" term="%22Beerens%2C+Koen%22">Beerens, Koen</searchLink><relatesTo>2</relatesTo><br /><searchLink fieldCode="AR" term="%22Merkaš%2C+Magdalena%22">Merkaš, Magdalena</searchLink><relatesTo>4</relatesTo><br /><searchLink fieldCode="AR" term="%22Pavkov-Keller%2C+Tea%22">Pavkov-Keller, Tea</searchLink><relatesTo>5</relatesTo><br /><searchLink fieldCode="AR" term="%22Desmet%2C+Tom%22">Desmet, Tom</searchLink><relatesTo>2,6</relatesTo><br /><searchLink fieldCode="AR" term="%22Nidetzky%2C+Bernd%22">Nidetzky, Bernd</searchLink><relatesTo>1,6</relatesTo><i> bernd.nidetzky@tugraz.at</i>
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  Data: <searchLink fieldCode="JN" term="%22Journal+of+Biological+Chemistry%22">Journal of Biological Chemistry</searchLink>. May2026, Vol. 302 Issue 5, p1-17. 17p.
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  Data: <searchLink fieldCode="DE" term="%22Epimerases%22">Epimerases</searchLink><br /><searchLink fieldCode="DE" term="%22Epimerization%22">Epimerization</searchLink><br /><searchLink fieldCode="DE" term="%22Molecular+dynamics%22">Molecular dynamics</searchLink><br /><searchLink fieldCode="DE" term="%22Enzyme+specificity%22">Enzyme specificity</searchLink><br /><searchLink fieldCode="DE" term="%22Crystal+structure%22">Crystal structure</searchLink><br /><searchLink fieldCode="DE" term="%22Pyrimidine+nucleotides%22">Pyrimidine nucleotides</searchLink><br /><searchLink fieldCode="DE" term="%22Catalysis%22">Catalysis</searchLink>
– Name: Abstract
  Label: Abstract
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  Data: The CDP–tyvelose 2-epimerase from Thermodesulfatator atlanticus (TaTyvE) catalyzes the C-2 epimerization of CDP–glucose to CDP–mannose. The enzyme uses NADdependent oxidation–reduction to achieve C-2 configurational inversion of the substrate. Here, we report the 2.60-Å crystal structure of tetrameric TaTyvE with NAD+ bound in all subunits and CDP bound in one (Protein Data Bank code: 9RL0). Binding of CDP orders the Gly197–Trp207 loop, closing over the sugar binding pocket. The active site of the ternary complex is well preorganized, with only moderate induced-fit conformational changes. Molecular dynamics simulations and site-directed mutagenesis suggest that TaTyvE employs a dual catalytic architecture to control substrate specificity. Asn125, within the TNK segment (Thr124–Asn125–Lys126), promotes sampling of catalytically plausible glucose conformations. The VAM segment (Val83–Ala84–Met85) permits broader conformational flexibility for mannose through backbone contacts. This is supported by a modest ~ 12-fold activity reduction in the Q205A variant. In contrast, N125A abolishes activity. Substrate analogs featuring deoxygenation, stereoinversion, or fluorination at the C-4 were synthesized to examine the role of the sugar C4–OH. Simulations indicated that the C4–OH interacts with Val83, Asn125, and Gln205. Activity was lost with CDP–4-deoxy-glucose and minimally recovered with CDP–4-fluoro-glucose. Removing the C5- hydroxymethyl group to restrict substrate positioning flexibility enhanced the reaction rate. Overall, these results highlight the important interplay of structural preorganization and conformational flexibility in TaTyvE for enzyme activity and specificity in the C-2 epimerization of CDP–glucose. [ABSTRACT FROM AUTHOR]
– Name: AbstractSuppliedCopyright
  Label:
  Group: Ab
  Data: <i>Copyright of Journal of Biological Chemistry is the property of Elsevier B.V. and its content may not be copied or emailed to multiple sites without the copyright holder's express written permission. Additionally, content may not be used with any artificial intelligence tools or machine learning technologies. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract.</i> (Copyright applies to all Abstracts.)
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RecordInfo BibRecord:
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      – Type: doi
        Value: 10.1016/j.jbc.2026.111384
    Languages:
      – Code: eng
        Text: English
    PhysicalDescription:
      Pagination:
        PageCount: 17
        StartPage: 1
    Subjects:
      – SubjectFull: Epimerases
        Type: general
      – SubjectFull: Epimerization
        Type: general
      – SubjectFull: Molecular dynamics
        Type: general
      – SubjectFull: Enzyme specificity
        Type: general
      – SubjectFull: Crystal structure
        Type: general
      – SubjectFull: Pyrimidine nucleotides
        Type: general
      – SubjectFull: Catalysis
        Type: general
    Titles:
      – TitleFull: A dual catalytic architecture promotes C-2 stereoinversion of CDP–glucose by the CDP–tyvelose 2-epimerase from Thermodesulfatator atlanticus.
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            NameFull: Rapp, Christian
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            – D: 01
              M: 05
              Text: May2026
              Type: published
              Y: 2026
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              Value: 302
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              Value: 5
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