title: Differential role of NADP+ and NADPH in the activity and structure of GDP-D-mannose 4,6-dehydratase from two chlorella viruses.
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.
authors.affiliation: Department of Experimental Medicine, University of Genova and Center of Excellence for Biomedical Research, Viale Benedetto XV, 1, 16132, Genova, Italy.
authors.forename: Floriana
authors.initials: F
authors.lastname: Fruscione
authors.affiliation: Department of Experimental Medicine, University of Genova and Center of Excellence for Biomedical Research, Viale Benedetto XV, 1, 16132, Genova, Italy.
authors.forename: Laura
authors.initials: L
authors.lastname: Sturla
authors.affiliation: Department of Biology, Nebraska Wesleyan University, Lincoln, Nebraska 68504-2794.
authors.forename: Garry
authors.initials: G
authors.lastname: Duncan
authors.affiliation: Department of Plant Pathology and Nebraska Center for Virology, University of Nebraska, Lincoln, Nebraska 68583-0722.
authors.forename: James L
authors.initials: JL
authors.lastname: Van Etten
authors.affiliation: Department of Plant Pathology and Nebraska Center for Virology, University of Nebraska, Lincoln, Nebraska 68583-0722.
authors.forename: Paola
authors.initials: P
authors.lastname: Valbuzzi
authors.affiliation: Department of Experimental Medicine, University of Genova and Center of Excellence for Biomedical Research, Viale Benedetto XV, 1, 16132, Genova, Italy.
authors.forename: Antonio
authors.initials: A
authors.lastname: De Flora
authors.affiliation: Department of Experimental Medicine, University of Genova and Center of Excellence for Biomedical Research, Viale Benedetto XV, 1, 16132, Genova, Italy.
authors.forename: Eleonora
authors.initials: E
authors.lastname: Di Zanni
authors.affiliation: Department of Experimental Medicine, University of Genova and Center of Excellence for Biomedical Research, Viale Benedetto XV, 1, 16132, Genova, Italy. Electronic address: tonetti@unige.it.
authors.forename: Michela
authors.initials: M
authors.lastname: Tonetti
mesh_terms: D002708:Chlorella / Q000821:virology*; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D006154:Guanosine Diphosphate Fucose / Q000737:chemistry / Q000378:metabolism; D006155:Guanosine Diphosphate Mannose / Q000737:chemistry / Q000378:metabolism; D006836:Hydro-Lyases / Q000737:chemistry / Q000145:classification / Q000378:metabolism*; D015394:Molecular Structure; D009249:NADP / Q000378:metabolism*; D017862:Phycodnaviridae / Q000378:metabolism*; D010802:Phylogeny; D013050:Spectrometry, Fluorescence / Q000379:methods; D014764:Viral Proteins / Q000737:chemistry / Q000145:classification / Q000378:metabolism*
chemical_list: D014764:Viral Proteins; D006154:Guanosine Diphosphate Fucose; D006155:Guanosine Diphosphate Mannose; D009249:NADP; D006836:Hydro-Lyases; C044971:GDPmannose 4,6-dehydratase