Abstract
Haloferax mediterranei glucose dehydrogenase (EC 1.1.1.47) belongs to the medium-chain alcohol dehydrogenase superfamily and requires zinc for catalysis. In the majority of these family members, the catalytic zinc is tetrahedrally coordinated by the side chains of a cysteine, a histidine, a cysteine or glutamate and a water molecule. In H. mediterranei glucose dehydrogenase, sequence analysis indicates that the zinc coordination is different, with the invariant cysteine replaced by an aspartate residue. In order to analyse the significance of this replacement and to contribute to an understanding of the role of the metal ion in catalysis, a range of binary and ternary complexes of the wild-type and a D38C mutant protein have been crystallized. For most of the complexes, crystals belonging to space group I222 were obtained using sodium/potassium citrate as a precipitant. However, for the binary and non-productive ternary complexes with NADPH/Zn, it was necessary to replace the citrate with 2-methyl-2,4-pentanediol. Despite the radical change in conditions, the crystals thus formed were isomorphous.
Highlights
Halophilic archaea are found in highly saline environments such as natural salt lakes or saltern pools
Sequence analysis has shown that Glucose dehydrogenase (GlcDH) belongs to the zinc-dependent medium-chain alcohol dehydrogenase (MDR) superfamily (Bonete et al, 1996; Pire et al, 2001)
The crystallization of recombinant GlcDH in the presence of NADP+ has been reported under conditions which closely mimic those experienced by the enzyme in the cell of the halophile (Ferrer et al, 2001); more recently, the structure has been determined to 1.6 Aresolution (Britton et al, 2005)
Summary
Halophilic archaea are found in highly saline environments such as natural salt lakes or saltern pools. The structure of H. mediterranei GlcDH has shown that there is an additional difference, with an aspartate residue (Asp38) occurring in a structurally equivalent position to the highly conserved first cysteine of this motif (Britton et al, 2005) This is a sequence change rarely seen in the MDR family, but that has been observed in the sequence of other halophilic glucose dehydrogenases [for example, the Halobacterium sp (Ng et al, 2000) and Haloferax volcanii GlcDHs; http://zdna2.umbi.umd.edu] and raises the intriguing question as to whether the presence of this aspartate residue in the active site is a halophilic adaptation. We report the preliminary crystallographic analysis of these various enzyme–substrate complexes
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