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Structural characterization of the thermostable Bradyrhizobium japonicum d ‐sorbitol dehydrogenase
Author(s) -
Fredslund Folmer,
Otten Harm,
Gemperlein Sabrina,
Poulsen Jens-Christian N.,
Carius Yvonne,
Kohring Gert-Wieland,
Lo Leggio Leila
Publication year - 2016
Publication title -
acta crystallographica section f
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.572
H-Index - 37
ISSN - 2053-230X
DOI - 10.1107/s2053230x16016927
Subject(s) - thermostability , tetramer , bradyrhizobium japonicum , sorbitol dehydrogenase , molecular replacement , crystallography , sorbitol , chemistry , dehydrogenase , rhodobacter sphaeroides , biochemistry , stereochemistry , enzyme , biology , bacteria , rhizobiaceae , photosynthesis , symbiosis , genetics
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 T m for 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.