Structure ofEscherichia coliglutamate decarboxylase (GADα) in complex with glutarate at 2.05 Å resolution

Abstract

Glutamate decarboxylase (GAD) is a pyridoxal enzyme that catalyzes the conversion of L-glutamate into gamma-aminobutyric acid and carbon dioxide. The Escherichia coli enzyme exists as two isozymes, referred to as GADalpha and GADbeta. Crystals of the complex of the recombinant isozyme GADalpha with glutarate as a substrate analogue were grown in space group R3, with unit-cell parameters a = b = 117.1, c = 196.4 angstroms. The structure of the enzyme was solved by the molecular-replacement method and refined at 2.05 angstroms resolution to an R factor of 15.1% (R(free) = 19.9%). The asymmetric unit contains a dimer consisting of two subunits of the enzyme related by a noncrystallographic twofold axis which is perpendicular to and intersects a crystallographic threefold axis. The dimers are related by a crystallographic threefold axis to form a hexamer. The active site of each subunit is formed by residues of the large domains of both subunits of the dimer. The coenzyme pyridoxal phosphate (PLP) forms an aldimine bond with Lys276. The glutarate molecule bound in the active site of the enzyme adopts two conformations with equal occupancies. One of the two carboxy groups of the glutarate occupies the same position in both conformations and forms hydrogen bonds with the N atom of the main chain of Phe63 and the side chain of Thr62 of one subunit and the side chains of Asp86 and Asn83 of the adjacent subunit of the dimer. Apparently, it is in this position that the distal carboxy group of the substrate would be bound by the enzyme, thus providing recognition of glutamic acid by the enzyme.

A Common Structural Basis for pH- and Calmodulin-mediated Regulation in Plant Glutamate Decarboxylase

“…The GadB residues Asn83 ⁎ †, Asp86 ⁎ †, Thr62 and Phe63 are predicted to be involved in binding of the distal carboxylate of the substrate glutamate, based on the crystal structure of the bacterial isoform GadA in complex with glutarate. 13 Gad1 residues Asn83 ⁎ , Asp86 ⁎ and Phe63 are conserved and found in conformations similar to those in GadB. Thr62 in GadB is replaced by serine in Gad1 (Ser62), although its side-chain hydroxyl group is at the same position, ready to act as a hydrogen donor upon substrate binding (Fig.…”

The E. coli sirtuin CobB shows no preference for enzymatic and nonenzymatic lysine acetylation substrate sites

“…The helix on which Lys‐4 resides helps to stabilize the hexamer (Dutyshev et al. ). To our knowledge, the role of Lys‐4 in GadA has not been described and thus the biological effect of acetylating this residue remains unknown.…”

Glutamate decarboxylase‐dependent acid resistance in orally acquired bacteria: function, distribution and biomedical implications of the gadBC operon

“…B), was reported in 2003 (Capitani et al ., ). This was the first structure of a glutamate decarboxylase to be released, followed shortly after by the release of the structure of E. coli GadA in complex with glutarate, a substrate analogue (Dutyshev et al ., ). More recently, the structures of the two isoforms of human GAD (GAD65 and GAD67) and of the Arabidopsis thaliana Gad1 isoform were disclosed (Fenalti et al ., ; Gut et al ., ).…”