PDBsum entry 1d4f

Hydrolase

PDB id: 1d4f

Contents

Protein chains

430 a.a. *

Ligands

NAD ×4

ADN ×4

Waters ×473

* Residue conservation analysis

References listed in PDB file

Key reference

Title

Effects of site-Directed mutagenesis on structure and function of recombinant rat liver s-Adenosylhomocysteine hydrolase. Crystal structure of d244e mutant enzyme.

Authors

J.Komoto, Y.Huang, T.Gomi, H.Ogawa, Y.Takata, M.Fujioka, F.Takusagawa.

Ref.

J Biol Chem, 2000, 275, 32147-32156. [DOI no: 10.1074/jbc.M003725200]

PubMed id

10913437

Abstract

A site-directed mutagenesis, D244E, of S-adenosylhomocysteine hydrolase (AdoHcyase) changes drastically the nature of the protein, especially the NAD(+) binding affinity. The mutant enzyme contained NADH rather than NAD(+) (Gomi, T., Takata, Y., Date, T., Fujioka, M., Aksamit, R. R., Backlund, P. S., and Cantoni, G. L. (1990) J. Biol. Chem. 265, 16102-16107). In contrast to the site-directed mutagenesis study, the crystal structures of human and rat AdoHcyase recently determined have shown that the carboxyl group of Asp-244 points in a direction opposite to the bound NAD molecule and does not participate in any hydrogen bonds with the NAD molecule. To explain the discrepancy between the mutagenesis study and the x-ray studies, we have determined the crystal structure of the recombinant rat-liver D244E mutant enzyme to 2.8-A resolution. The D244E mutation changes the enzyme structure from the open to the closed conformation by means of a approximately 17 degrees rotation of the individual catalytic domains around the molecular hinge sections. The D244E mutation shifts the catalytic reaction from a reversible to an irreversible fashion. The large affinity difference between NAD(+) and NADH is mainly due to the enzyme conformation, but not to the binding-site geometry; an NAD(+) in the open conformation is readily released from the enzyme, whereas an NADH in the closed conformation is trapped and cannot leave the enzyme. A catalytic mechanism of AdoHcyase has been proposed on the basis of the crystal structures of the wild-type and D244E enzymes.

Figure 2.
Fig. 2. Ribbon drawing of a single subunit of AdoHcyase showing three domains: catalytic domain, NAD^+-binding domain, and C-terminal domain. The C-terminal domain from the subunit B is illustrated with white. The bound NAD molecule and Ado* are illustrated by ball-and-stick modes. A, WT structure (open conformation); B, D244E structure (closed conformation).

Figure 7.
Fig. 7. A movement of the section of C-terminal domain (residues: 420B-430B) of subunit B. A, WT structure (open conformation); B, D244E structure (closed conformation). The NAD-binding domains are aligned by a least-squares method.

The above figures are reprinted by permission from the ASBMB: J Biol Chem (2000, 275, 32147-32156) copyright 2000.