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Protein Science (2004), 13:3161-3171. Published by Cold Spring Harbor Laboratory Press. Copyright © 2004 The Protein Society
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Structural and functional analysis of the gpsA gene product of Archaeoglobus fulgidus: A glycerol-3-phosphate dehydrogenase with an unusual NADP+ preference

Shin-Ichi Sakasegawa1,3,4, Christoph H. Hagemeier1,3, Rudolf K. Thauer1, Lars-O. Essen2 and Seigo Shima1

1 Max-Planck-Institut für terrestrische Mikrobiologie and Laboratorium für Mikrobiologie, Fachbereich Biologie, and 2 Fachbereich Chemie, Philipps-Universität, Marburg, Germany

(RECEIVED July 9, 2004; FINAL REVISION August 17, 2004; ACCEPTED August 17, 2004)

NAD+-dependent glycerol-3-phosphate dehydrogenase (G3PDH) is generally absent in archaea, because archaea, unlike eukaryotes and eubacteria, utilize glycerol-1-phosphate instead of glycerol-3-phosphate for the biosynthesis of membrane lipids. Surprisingly, the genome of the hyperthermophilic archaeon Archaeoglobus fulgidus comprises a G3PDH ortholog, gpsA, most likely due to horizontal gene transfer from a eubacterial organism. Biochemical characterization proved G3PDH-like activity of the recombinant gpsA gene product. However, unlike other G3PDHs, the up to 85°C thermostable A. fulgidus G3PDH exerted a 15-fold preference for NADPH over NADH. The A. fulgidus G3PDH bears the hallmarks of adaptation to halotolerance and thermophilicity, because its 1.7-Å crystal structure showed a high surface density for negative charges and 10 additional intramolecular salt bridges compared to a mesophilic G3PDH structure. Whereas all amino acid residues required for dihydroxyacetone phosphate binding and reductive catalysis are highly conserved, the binding site for the adenine moiety of the NAD(P) cosubstrate shows a structural variation that reflects the observed NADPH preference, for example, by a putative salt bridge between R49 and the 2'-phosphate.

Keywords: glycerol-3-phosphate dehydrogenase; crystal structure; diglycerol phosphate; Archaeoglobus fulgidus; Leishmania mexicana; thermophilic enzymes

Article and publication are at http://www.proteinscience.org/cgi/doi/10.1110/ps.04980304.

Reprint requests to: Lars-O. Essen, Fachbereich Chemie, Philipps Universität, Hans-Meerwein-Strasse, 35032 Marburg, Germany; e-mail: essen{at}chemie.uni-marburg.de; fax: +49-6421-28-22191; or Seigo Shima, Max-Planck-Institut für terrestrische Mikrobiologie and Laboratorium für Mikrobiologie, Fachbereich Biologie, Philipps-Universität, Karl-von-Frisch-Strasse, 35043 Marburg, Germany; e-mail: shima{at}staff.uni-mar-burg.de; fax: +49-6421-178199.


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