Differential role of NADP+ and NADPH in the activity and structure of GDP-D-mannose 4,6-dehydratase from two chlorella viruses

Floriana Fruscione, Laura Sturla, Garry Duncan, James L. Van Etten, Paola Valbuzzi, Antonio De Flora, Eleonora Di Zanni, Michela Tonetti

Research output: Contribution to journalArticlepeer-review

25 Scopus citations

Abstract

GDP-D-mannose 4,6-dehydratase (GMD) is a key enzyme involved in the synthesis of 6-deoxyhexoses in prokaryotes and eukaryotes. Paramecium bursaria chlorella virus-1 (PBCV-1) encodes a functional GMD, which is unique among characterized GMDs because it also has a strong stereospecific NADPH-dependent reductase activity leading to GDP-D-rhamnose formation (Tonetti, M., Zanardi, D., Gurnon, J., Fruscione, F., Armirotti, A., Damonte, G., Sturla, L., De Flora, A., and Van Etten, J.L. (2003) J. Biol. Chem. 278, 21559-21565). In the present study we characterized a recombinant GMD encoded by another chlorella virus, Acanthocystis turfacea chlorella virus 1 (ATCV-1), demonstrating that it has the expected dehydratase activity. However, it also displayed significant differences when compared with PBCV-1 GMD. In particular, ATCV-1 GMD lacks the reductase activity present in the PBCV-1 enzyme. Using recombinant PBCV-1 and ATCV-1 GMDs, we determined that the enzymatically active proteins contain tightly bound NADPH and that NADPH is essential for maintaining the oligomerization status as well as for the stabilization and function of both enzymes. Phylogenetic analysis indicates that PBCV-1 GMD is the most evolutionary diverged of the GMDs. We conclude that this high degree of divergence was the result of the selection pressures that led to the acquisition of new reductase activity to synthesize GDP-D-rhamnose while maintaining the dehydratase activity in order to continue to synthesize GDP-L-fucose.

Original languageEnglish (US)
Pages (from-to)184-193
Number of pages10
JournalJournal of Biological Chemistry
Volume283
Issue number1
DOIs
StatePublished - Jan 4 2008

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Fingerprint Dive into the research topics of 'Differential role of NADP<sup>+</sup> and NADPH in the activity and structure of GDP-D-mannose 4,6-dehydratase from two chlorella viruses'. Together they form a unique fingerprint.

Cite this