X-ray crystal structure and small-angle X-ray scattering of sheep liver sorbitol dehydrogenase

Abstract

The X-ray crystal structure of sheep liver sorbitol dehydrogenase (slSDH) has been determined using the crystal structure of human sorbitol dehydrogenase (hSDH) as a molecular-replacement model. slSDH crystallized in space group I222 with one monomer in the asymmetric unit. A conserved tetramer that superposes well with that seen in hSDH (despite belonging to a different space group) and obeying the 222 crystal symmetry is seen in slSDH. An acetate molecule is bound in the active site, coordinating to the active-site zinc through a water molecule. Glycerol, a substrate of slSDH, also occupies the substrate-binding pocket together with the acetate designed by nature to fit large polyol substrates. The substrate-binding pocket is seen to be in close proximity to the tetramer interface, which explains the need for the structural integrity of the tetramer for enzyme activity. Small-angle X-ray scattering was also used to identify the quaternary structure of the tetramer of slSDH in solution.

Enantioselective oxidation of galactitol 1-phosphate by galactitol-1-phosphate 5-dehydrogenase fromEscherichia coli

“…In this type of coordination where Zn 2+ forms four strong bonds to cysteine residues the metal does not normally dissociate under biological conditions (Harding et al, 2010), usually playing a role in stabilization of the tertiary structure. This structural metal is also observed in the crystal structures of the close homologues the l-threonine dehydrogenases from P. horikoshii (Ishikawa et al, 2007) and from T. thermophilus (PDB entry 2dfv) and whitefly ketose reductase (Banfield et al, 2001), but not in hSDH (Pauly et al, 2003), sheep liver sorbitol dehydrogenase (Yennawar et al, 2011) or l-threonine dehydrogenase from T. kodakaraensis (Bowyer et al, 2009). This variability in the structural metal-binding site correlates well with the variation in the total zinc content observed in general for other more distant ADHs (Bogin et al, 1997;Korkhin et al, 1998;Sulzenbacher, Alvarez et al, 2004).…”

“…The structures of GPDH with Tris or glycerol within the active site reveal features that may be relevant to the wider MDR superfamily, in particular aspects related to the pair of acidic residues close to the zinc centre (Glu60 and Glu144 in GPDH). The acidic residue Glu144 is conserved among the PDHs (Jeffery & Jö rnvall, 1988) and is not a ligand of the Zn 2+ , but coordinates the metal via a water molecule whose conserved character in PDHs [human (Pauly et al, 2003) and sheep (Yennawar et al, 2011) sorbitol dehydrogenases and whitefly ketose reductase (Banfield et al, 2001)] suggests that it may play a role in catalysis (Klimacek et al, 2007). In GPDH this solvent molecule also interacts with the accompanying acidic residue Glu60, which similarly to Glu144 does not coordinate the Zn 2+ .…”

Polyol specificity of recombinant Arabidopsis thaliana sorbitol dehydrogenase studied by enzyme kinetics and in silico modeling

“…Thus, a catalytic mechanism whereby the catalytic zinc changes from a tetrahedric coordination in the absence of substrates to a pentahedric geometry where hydroxyls 1 and 2 of sorbitol become part of the coordination sphere, was proposed ( Pauly et al, 2003 ). More recently, an SDH from the liver of sheep ( Ovis aries ) has been crystallized (PDB 3QE3, Yennawar et al, 2011 ) in the presence of catalytic zinc. In this structure, an acetate molecule is observed close to the coordination sphere of the zinc atom, and a glycerol molecule is bound through hydrogen bonds with arginine, tyrosine, and glutamic acid residues of the binding pocket.…”

High-throughput biological small-angle X-ray scattering with a robotically loaded capillary cell

“…Reduced effects of radiation damage due to flow have also proven advantageous to users. A number of studies have now appeared in the literature utilizing this system (Byrnes & Sondermann, 2011;Yennawar et al, 2011;Jiang et al, 2011).…”