Photon Factory Activity Report 2004 #22 Part B (2005)
Biological Science

10C/2003G144

Low resolution modeled structure of inactive recombinant glutamate
dehydrogenase from Pyrobaculum islandicum by ab initio method
Shuichiro GODA1, Yoshimi NISHIKAWA1, Masaki
KOJIMA2, Haruhiko
SAKURABA1, Toshihisa
1*3
OHSHIMA , Yuzuru HIRAGI
1TheUniversityof Tokushima, Tokushima 770-8506, Japan.
2
Tokyo University
ofPharmacy
and Life Science, Hachioji, Tokyo 192-0392, Japan
3
Kansai Medical University, Hirakata, Osaka 573-1136, Japan.
Introduction
The specific activity of recombinant Pyrobaculum
islandicum glutamate dehydrogenase (GDH) expressed inEscherichia coli is much lower than that of the native
GDH. However, upon heating the inactive recombinant
GDH (iGDH) at 90˚C, the activity increases to a levelcomparable to that of the native GDH. Small-angle X-ray
scattering (SAXS) measurements revealed that the radiusof gyration of the hexameric iGDH was reduced to 47 Åfrom 55 Å by heat, though the molecular mass of theGDH was unchanged [1, 2]. Although the crystal
structure of activated GDH (aGDH) has been described
previously [3], the structure of the iGDH remains
unknown. We modeled the low-resolution structure of theiGDH in an ab initio manner from the scattering curve.
Materials and Methods
Recombinant GDH was expressed in E. coli.
Expression condition, purification procedure, SAXS
measurements and analysis were described elsewhere [1,2]. The modeling program, DAMMIN represents particle
shape as an ensemble of densely packed beads inside aspherical search volume with diameter Dmax [4].
Starting from a random distribution of beads, simulated
annealing is employed to find a compact configuration
minimizing the discrepancy χ between the experimental
and calculated scattering curves. Several independent
reconstructions were carried out by randomly
approximating the initial packing radii of the dummy
atoms (ro = 3-4 Å) to compute the average and the mostprobable models.
Results and Discussion
As DAMMIN permits modeling using symmetry
restrictions on the solution, the low-resolution structure of
the aGDH was restored by 32-point symmetry, likecrystal structure [3], and was well fitted to the crystalstructure (Fig. 1a,b). Then, structure of the iGDH wasmodeled no symmetry and 32-point symmetry (Fig. 1c,d).
The structure of iGDH restored using 32-point symmetry
differed substantially from that of the aGDH (Fig. 1c,d,top row), and appeared unlikely in terms of the subunit
construction. On the other hand, the overall structure
restored with no symmetry was similar to that of theaGDH (Fig. 1b,c). These results suggest that the nosymmetry structure is more feasible for iGDH. Thestructure of iGDH has two features that distinguish it236

Users' Report

Figure 1. Crystal and low-resolution structures ofGDH. (a) Crystal structure of aGDH. (b) Lowresolution structure of the aGDH with 32-point
symmetry. Low-resolution structures of the iGDH
with no symmetry (c) and 32-point symmetry (d).
from the aGDH. The first is its lack of molecularsymmetry. The aGDH has 32-point symmetry with one
3-fold axis passing through the two identical trimers,
which face one another yielding a whole molecule with acylindrical shape (Fig. 1a,b, bottom row) and one trimer
stacking on the second with a symmetrical interface (Fig.
1a,b, top row). On the other hand, the structure of theiGDH has no symmetry; consequently, no subunit from
one trimer superimposes on the corresponding subunit of
the other (Fig. 1c, top row). The second structural featureof the iGDH is that it is not cylindrical, but is instead
more spread out, especially at the surface of the molecule(Fig. 1c, middle row). It thus appears that not only isoligomerization of GDH necessary for enzymatic activity,
but the proper arrangement of the subunits is alsoessential.
References[1] S. Goda et al., PF activity reprots 252 (2002).
[2] Y. Nishikawa et al., PF activity reports 236 (2003).
[3] M. W. Bhuiya, et al., J. Mol. Biol. 345, 325-337(2005).
[4] D. I. Svergun, Biophys. J. 76, 2879-2886 (1999).
* yhiragi@yahoo.co.jp