Glutamate decarboxylase 1(GAD) / cofactor PLP complexed
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.
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).
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.
The differences between two active sites are described below.
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.
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.
Protein Data Bank (PDB)
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.
author: Jun-ichi Ito