RSS

PDB:2OKJ

Protein Name

Glutamate decarboxylase 1(GAD) / cofactor PLP complexed

Species

human

Biological Context

Glutamate decarboxylase(GAD) is an enzyme that synthesizes inhibitory neurotransmitter γ-aminobutyric acid(GABA) by glutamate decarboxylation. (Fig.1). With docking to a cofactor pyridoxal phosphate(PLP), GAD becomes activated holoenzyme and can express the function. In mammals, GAD exists in two isoforms(GAD67 and GAD65) encoded by two different genes. GAD67 is constantly active and is responsible for basal GABA production. In contrast, GAD65 is temporarily activated in response to the demand for extra GABA in neurotransmission. For investigate the structural basis for the functional differences between the two GAD isoforms, author have determined the crystal structures of GAD67 and GAD65.

1
(Fig.1) Two passages of glutamate decarboxylation reaction by GAD67

(1), A cofactor PLP bind to Lys405 at the active site of GAD67. Then GAD67 becomes active holoenzyme.
(2), Quinoid intermediate is formed by the binding of grutamate to Lys405-PLP complex.

The reaction is divided into two passages from here.

(3), If quinoid is protonated at Cα site.
Quinoid is reduced to Lys405-PLP with releasing GABA. GAD remains active~ holoenzyme.

(3'), If quinoid is protonated at C4' site.
Pyridoxamine 5'-phosphate (PMP) and Succinic semialdehyde(SSA) are released. Because the cofactor PLP is separated as the PMP,
GAD becomes inactive apoenzyme.
  • (Supplementary)
    • Molecular weights of GAD67 and GAD65 are 67kDa and 65 kDa respectively.
    • GAD67 and GAD65 are also known as GAD1 and GAD2.
    • Many PLP-dependent enzymes exist besides GAD. They catalyze various biological reactions.
      • Transamination(PDB:1DAA)
      • Aldol cleavage
      • Racemization

Structure Description

2okj2okj_x2okj_y

Author determined a crystal structure of GAD67(residues 90-594) that is enzymatically active with PLP binding. GAD67 is a homodimer with each monomeric unit comprises three domains [ N-terminal(residues 93-196), PLP-binding(197-472) and C-terminal(473-593)]. (Fig.2).

2
(Fig.2) Dimer of GAD67

Two active sites are located in the interface of two PLP-binding domains. (Fig.3). Lys405 in each active site is bound to a PLP. Furthermore, each active site is substantially covered by an extended loop(residues 432-442: termed the "catalytic loop"). Remarkably, Tyr434 of the catalytic loop is close to GABA.

3
(Fig.3) Two active sites
The direction of Tyr434 in the active site A is different to it in the active site B

The differences between two active sites are described below.

  • In active site A
    • GABA is separated from Lys405-PLP complex and be isolated.
    • The head(hydroxyl group) of Tyr434 is 2.8Å from Cα site of a quinoid(PLP-GABA complex).
  • In active site B
    • GABA bind to Lys405-PLP complex and forms a quinoid.
    • The head of Tyr434 is over 6.0Å from Cα site of the quinoid.

These observations suggest Tyr434 directly protonates the Cα of quinoid and releasing GABA. Thus, mobility of Tyr434 facilitate substrate(grutamate) ingress and product(GABA) egress from the active site. When Tyr434 was replaced by other residue, GAD67 stopped to produce GABA. This means the residue is essential for catalytic activity.

Author also determined a crystal structure of GAD65(residues 84-585) bound to PLP. (The region of the catalytic loop was disordered, so could not been determined). GAD65 is 71% sequence identical to GAD67 and the structure is almost the same to GAD67. However, several residues in contact with the catalytic loop of GAD67 are different to GAD65. For example, Tyr292 in GAD67 is substituted by a phenylalanine(Phe283) in GAD65.
Author conducted interchange test described below.

Interchange Activity
GAD65 Replace Phe283 by a Tyr Slightly increase
GAD65 Insert catalytic loop of GAD67 The same
GAD65 Replace Phe283 by a Tyr & Insert catalytic loop of GAD67 Increase to about 200%
GAD67 Replace Tyr292 by a Phe Slightly decrease
GAD67 Insert catalytic loop of GAD65 About 25% decrease
GAD67 Replace Tyr292 by a Phe & Insert catalytic loop of GAD65 About 60% decrease

Thus, the functional differences between the two isoforms results from both the catalytic loop and the residues against this region.

They can be summarized as follows.
For GAD67, the continuous presence of Tyr434 in the active site favors protonation of the Cα site and uninterrupted GABA production.
On the other hand, the catalytic loop of GAD65 is mobile. The transient absence of the catalytic tyrosine allows protonation at the C4' site of quinoid and inactivation of GAD65. This leads to intermittent GABA production of GAD65.

Protein Data Bank (PDB)

References

Source

Fenalti, G. Law, R.H.P. Buckle, A.M. Langendorf, C. Tuck, K. Rosado, C.J. Faux, N.G. Mahmood, K. Hampe, C.S. Banga, J.P. Wilce, M. Schmidberger, J. Rossjohn, J. El-Kabbani, O. Pike, R.N. Smith, A.I. Mackay, I.R. Rowley, M.J. Whisstock, J.C.; "GABA production by glutamic acid decarboxylase is regulated by a dynamic catalytic loop."; Nat.Struct.Mol.Biol.; (2007) 14:280-286 PubMed:17384644.

Others

author: Jun-ichi Ito


Japanese version:PDB:2OKJ