Structural characterization of the thermostable Bradyrhizobium japonicumD-sorbitol dehydrogenase.
Bradyrhizobium japonicum sorbitol dehydrogenase is NADH-dependent and is active at elevated temperatures. The best substrate is D-glucitol (a synonym for D-sorbitol), although L-glucitol is also accepted, giving it particular potential in industrial applications. Crystallization led to a hexagonal crystal form, with crystals diffracting to 2.9 Å resolution. In attempts to phase the data, a molecular-replacement solution based upon PDB entry 4nbu (33% identical in sequence to the target) was found. The solution contained one molecule in the asymmetric unit, but a tetramer similar to that found in other short-chain dehydrogenases, including the search model, could be reconstructed by applying crystallographic symmetry operations. The active site contains electron density consistent with D-glucitol and phosphate, but there was not clear evidence for the binding of NADH. In a search for the features that determine the thermostability of the enzyme, the Tmfor the orthologue from Rhodobacter sphaeroides, for which the structure was already known, was also determined, and this enzyme proved to be considerably less thermostable. A continuous β-sheet is formed between two monomers in the tetramer of the B. japonicum enzyme, a feature not generally shared by short-chain dehydrogenases, and which may contribute to thermostability, as may an increased Pro/Gly ratio.
Department of Chemistry, University of Copenhagen, Universitetsparken 5, DK-2100 Copenhagen, Denmark.
Folmer
F
Fredslund
Department of Chemistry, University of Copenhagen, Universitetsparken 5, DK-2100 Copenhagen, Denmark.
Harm
H
Otten
Microbiology, Saarland University, Campus Building A1.5, Saarbrücken, D-66123 Saarland, Germany.
Sabrina
S
Gemperlein
Department of Chemistry, University of Copenhagen, Universitetsparken 5, DK-2100 Copenhagen, Denmark.
Jens Christian N
JC
Poulsen
Department of Structural Biology, ZHMB, Saarland University, Building 60, D-66421 Homburg, Germany.
Yvonne
Y
Carius
Microbiology, Saarland University, Campus Building A1.5, Saarbrücken, D-66123 Saarland, Germany.
Gert Wieland
GW
Kohring
Department of Chemistry, University of Copenhagen, Universitetsparken 5, DK-2100 Copenhagen, Denmark.
Leila
L
Lo Leggio
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D020369:Bradyrhizobium / Q000737:chemistry* / Q000201:enzymology; D020134:Catalytic Domain; D003001:Cloning, Molecular; D018360:Crystallography, X-Ray; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D006358:Hot Temperature; D007064:L-Iditol 2-Dehydrogenase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D010957:Plasmids / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding; D000072756:Protein Conformation, alpha-Helical; D000072757:Protein Conformation, beta-Strand; D055503:Protein Multimerization; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012242:Rhodobacter sphaeroides / Q000737:chemistry / Q000201:enzymology; D013012:Sorbitol / Q000737:chemistry* / Q000378:metabolism; D013379:Substrate Specificity; D013816:Thermodynamics
D001426:Bacterial Proteins; D011994:Recombinant Proteins; D013012:Sorbitol; D007064:L-Iditol 2-Dehydrogenase
glucitol; sorbitol dehydrogenase; thermostability