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

Yennawar H, Møller M, Gillilan R, and Yennawar N (2011) Acta Crystallogr D Biol Crystallogr. 67(5): 440-6

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.

Materials/Methods

X-Ray Crystal Structure

Sheep liver SDH (slSDH) was purchased from Sigma and used with no further purification. The enzyme was crystallized using the nonphotochemical laser-induced nucleation technique with the commercially available PEG/Ion screen from Hampton Research. This crystallization screening method has been described by Yennawar et al. (2010). Crystals grown at pH 7.0 from 0.2 M lithium acetate dihydrate and 20% polyethylene glycol 3350 were used for data collection. The crystals were soaked for 10 min with 10% glycerol in the mother liquor before cryofreezing to 93 K. X-ray diffraction was observed to a resolution of 1.8 Å. The diffraction data were collected at the home laboratory (Penn State) on a Rigaku MicroMax-007 rotating-anode generator equipped with a Saturn 944+ CCD detector and an X-stream 2000 for cryocooling.

SAXS data collection

slSDH solutions at concentrations of 20, 16 and 10 mg ml-1 were prepared at each of the pH values 7.0, 9.4, 9.6, 10.4 and 10.6 used for the SAXS analysis. Buffer containing 0.2 M glycine-NaOH was used for pH 9.4, 9.6, 10.4 and 10.6, while 0.1M HEPES was used for pH 7.0. The protein solution was centrifuged at 14 000 rev min-1 for 10 min prior to data collection. SAXS data were collected on CHESS beamline F2 at 9.881 keV (1.2563 Å, the tantalum edge). The X-ray beam was collimated to 250 x 250 µm and was centered on a 2 mm diameter vertical quartz capillary tube with 10 µm thick walls (Hampton Research, Aliso Viejo, California, USA). The capillary tube and full X-ray flight path, including beamstop, were kept in vacuo to eliminate air scatter. Sample plugs of approximately 15-20 µl were delivered from a 96-well plate to the capillary using a Hudson SOLO single-channel pipetting robot (Hudson Robotics Inc., Springfield, New Jersey, USA). To reduce radiation damage, the sample plugs were oscillated in the X-ray beam using a computer-controlled syringe pump (Aurora Biomed, Vancouver, Canada). Images were collected on a Quantum 1 CCD detector (Area Detector Systems Corporation, Poway, California, USA) with sequential 180 s exposures being used to assess possible radiation damage.

Yennawar et al. 2011 Figure 4
(a) SAXS envelopes superimposed on two candidate tetramers derived from crystal lattice symmetry. Tetramer 1 (top) is structurally analogous to the asymmetric unit of hSDH and is reasonably well contained by the experimental envelope. Tetramer 2 (bottom) is less compact and is not well contained by the envelope. (b) Computed versus experimental scattering for tetramer models. Tetramer 1 most closely matches the experimental data in the regionq < 0.1 Å.

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