GDP-altrose as novel product of GDP-mannose 3,5-epimerase: Revisiting its reaction mechanism.

GDP-mannose 3,5-epimerase (GM35E) catalyzes the double epimerization of GDP-mannose to yield GDP-l-galactose. GDP-l-gulose (C5-epimer) has previously been detected as a byproduct of this reaction, indicating that C3,5-epimerization occurs through an initial epimerization at C5. Given these products, GM35E constitutes a valuable bridge between d- and l-hexoses. In order to fully exploit this potential, the enzyme might be subjected to specificity engineering for which profound mechanistic insights are beneficial. Accordingly, this study further elucidated GM35E's reaction mechanism. For the first time, the production of the C3-epimer GDP-altrose was demonstrated, resulting in an adjustment of the acknowledged reaction mechanism. As GM35E converts GDP-mannose to GDP-l-gulose, GDP-altrose and GDP-l-galactose in a 72:4:4:20 ratio, this indicates that the enzyme does not discriminate between the C3 and C5 position as initial epimerization site. This was also confirmed by a structural investigation. Based on a mutational analysis of the active site, residues S115 and R281 were attributed a stabilizing function, which is believed to support the reactivation process of the catalytic residues. This paper eventually reflected on some engineering strategies that aim to change the enzyme towards a single specificity. Copyright © 2020 Elsevier B.V. All rights reserved.

Citation

Ophelia Gevaert, Stevie Van Overtveldt, Matthieu Da Costa, Koen Beerens, Tom Desmet. GDP-altrose as novel product of GDP-mannose 3,5-epimerase: Revisiting its reaction mechanism. International journal of biological macromolecules. 2020 Dec 15;165(Pt B):1862-1868

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PMID: 33075338

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