PDBsum entry 1ofe

Oxidoreductase

PDB id: 1ofe

Contents

Protein chains

1485 a.a. *

Ligands

FMN ×2

F3S ×2

AKG ×2

ONL ×2

Waters ×289

* Residue conservation analysis

References listed in PDB file

Key reference

Title

The active conformation of glutamate synthase and its binding to ferredoxin.

Authors

R.H.Van den heuvel, D.I.Svergun, M.V.Petoukhov, A.Coda, B.Curti, S.Ravasio, M.A.Vanoni, A.Mattevi.

Ref.

J Mol Biol, 2003, 330, 113-128. [DOI no: 10.1016/S0022-2836(03)00522-9]

PubMed id

12818206

Abstract

Glutamate synthases (GltS) are crucial enzymes in ammonia assimilation in plants and bacteria, where they catalyze the formation of two molecules of L-glutamate from L-glutamine and 2-oxoglutarate. The plant-type ferredoxin-dependent GltS and the functionally homologous alpha subunit of the bacterial NADPH-dependent GltS are complex four-domain monomeric enzymes of 140-165 kDa belonging to the NH(2)-terminal nucleophile family of amidotransferases. The enzymes function through the channeling of ammonia from the N-terminal amidotransferase domain to the FMN-binding domain. Here, we report the X-ray structure of the Synechocystis ferredoxin-dependent GltS with the substrate 2-oxoglutarate and the covalent inhibitor 5-oxo-L-norleucine bound in their physically distinct active sites solved using a new crystal form. The covalent Cys1-5-oxo-L-norleucine adduct mimics the glutamyl-thioester intermediate formed during L-glutamine hydrolysis. Moreover, we determined a high resolution structure of the GltS:2-oxoglutarate complex. These structures represent the enzyme in the active conformation. By comparing these structures with that of GltS alpha subunit and of related enzymes we propose a mechanism for enzyme self-regulation and ammonia channeling between the active sites. X-ray small-angle scattering experiments were performed on solutions containing GltS and its physiological electron donor ferredoxin (Fd). Using the structure of GltS and the newly determined crystal structure of Synechocystis Fd, the scattering experiments clearly showed that GltS forms an equimolar (1:1) complex with Fd. A fundamental consequence of this result is that two Fd molecules bind consecutively to Fd-GltS to yield the reduced FMN cofactor during catalysis.

Figure 1.
Figure 1. Schematic representation of the reaction catalyzed by GltS. The ammonia produced in the amidotransferase domain is added onto 2-OG in the FMN-binding domain.

Figure 5.
Figure 5. (A) Presentation of the Fd-GltS active site in the FMN-binding domain with bound 2-OG and a model of bound 2-iminoglutarate. 2-OG and 2-iminoglutarate are depicted as ball-and-stick in green and red, respectively. Hydrogen bonds between Lys972 and Gln978 and the carbonyl oxygen atom of 2-OG are indicated by broken green lines. Potential hydrogen bonds between Fd-GltS and 2-iminoglutarate are indicated by broken red lines. (B) Schematic representation of the role of Lys972 and Glu903 for the dual functionality of the active site in the FMN-binding domain. Lys972 fixes 2-OG in the proper conformation for ammonia addition and polarizes the carbonyl oxygen atom. Upon ammonia addition and water release, Glu903 anchors 2-iminoglutarate in the proper conformation for reduction by FMN.

The above figures are reprinted by permission from Elsevier: J Mol Biol (2003, 330, 113-128) copyright 2003.