The crystal structure of Pyrococcus furiosus ornithine carbamoyltransferase reveals a key role for oligomerization in enzyme stability at extremely high temperatures

Abstract

The Pyrococcus furiosus (PF) ornithine carbamoyltransferase (OTCase; EC 2.1.3.3) is an extremely heat-stable enzyme that maintains about 50% of its activity after heat treatment for 60 min at 100 degrees C. To understand the molecular basis of thermostability of this enzyme, we have determined its three-dimensional structure at a resolution of 2.7 A and compared it with the previously reported structures of OTCases isolated from mesophilic bacteria. Most OTCases investigated up to now are homotrimeric and devoid of allosteric properties. A striking exception is the catabolic OTCase from Pseudomonas aeruginosa, which is allosterically regulated and built up of four trimers disposed in a tetrahedral manner, an architecture that actually underlies the allostery of the enzyme. We now report that the thermostable PF OTCase (420 kDa) presents the same 23-point group symmetry. The enzyme displays Michaelis-Menten kinetics. A detailed comparison of the two enzymes suggests that, in OTCases, not only allostery but also thermophily was achieved through oligomerization of a trimer as a common catalytic motif. Thermal stabilization of the PF OTCase dodecamer is mainly the result of hydrophobic interfaces between trimers, at positions where allosteric binding sites have been identified in the allosteric enzyme. The present crystallographic analysis of PF OTCase provides a structural illustration that oligomerization can play a major role in extreme thermal stabilization.

Figure 1

(A) Molecular surface illustration of the dodecameric oligomer, where one trimer is represented in white and the three other trimers in red. The molecule has an approximate diameter of 130 Å. (B) Ribbons drawing of the OTCase monomer. Helices from the CP binding domain and H12 are in red; helices from the ornithine binding domain are in pink. Each domain is organized around a β pleated sheet of five parallel strands, respectively, from top to bottom: B3, B2, B4, B5, and B1 for the CP binding domain (deep blue) and B10, B9, B6, B7, and B8 for the ornithine binding domain (light blue). (C) Molecular surface illustration of a trimer viewed along its 3-fold symmetry axis. The three CP binding domains are in yellow and the ornithine binding domains in white. Figures were generated by using grasp (35) (A and C) and molscript (36) and raster3d (37) (B).

Figure 2

View of the interface between trimers located around a 3-fold symmetry axis at one top of the dodecamer. The three H1 helices from CP domains that belong to three monomers are shown in ribbons. (A) Interface in PF, mainly composed of the hydrophobic residues W21, M29, I32, W33, and I36. The salt bridges between K28 and E25 also are shown. (B) Interface in PA composed of positively charged residues: R21, R28, R32, and R146, constituting a binding site for negatively charged allosteric effectors. The difference in compactness between both enzymes also is illustrated by the increased packing in the PA OTCase of the three helices H1 around the 3-fold symmetry axis. Figures were generated by using molscript (36) and raster3d (37).

Figure 3

Stereo view of the Cα backbone of PF (blue) and PA (red) monomers, where the two CP binding domains have been superimposed. The ornithine binding domains differ in a rotation of 8°, resulting in a domain closure in PF OTCase. The CP binding domains are at the bottom. The figure was generated by using molscript (36).