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GABA production by glutamic acid decarboxylase is regulated by a dynamic catalytic loop


Gamma-aminobutyric acid (GABA) is synthesized by two isoforms of the pyridoxal 5′-phosphate–dependent enzyme glutamic acid decarboxylase (GAD65 and GAD67). GAD67 is constitutively active and is responsible for basal GABA production. In contrast, GAD65, an autoantigen in type I diabetes, is transiently activated in response to the demand for extra GABA in neurotransmission, and cycles between an active holo form and an inactive apo form. We have determined the crystal structures of N-terminal truncations of both GAD isoforms. The structure of GAD67 shows a tethered loop covering the active site, providing a catalytic environment that sustains GABA production. In contrast, the same catalytic loop is inherently mobile in GAD65. Kinetic studies suggest that mobility in the catalytic loop promotes a side reaction that results in cofactor release and GAD65 autoinactivation. These data reveal the molecular basis for regulation of GABA homeostasis.

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Figure 1: Reactions catalyzed by GAD and sequence alignment of both GAD isoforms.
Figure 2: Dimeric structure of GAD67 within the asymmetric unit of the crystal.
Figure 3: Structural comparisons between GAD67 and GAD65.
Figure 4: Comparison of GAD67 and GAD65 active sites, in stereo.
Figure 5: Inactivation of GAD in the presence of glutamate.

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J.C.W. is a National Health and Medical Research Council of Australia (NHMRC) Principal Research Fellow and a Monash University Senior Logan Fellow. A.M.B. and M.W. are NHMRC Senior Research Fellows. A.I.S. is an NHMRC Principal Research Fellow. J.R. is an Australian Research Council Federation Fellow. G.F. is a PhD scholar funded by the CAPES Foundation, subordinated to the Ministry of Education, Brazil. This work was supported by the NHMRC, the Australian Research Council and the State Government of Victoria (Australia). We thank the staff at IMCA-CAT (The Advanced Photon Source) for technical assistance, the Australian Synchrotron Research Program for support and M. Dunstone for critical reading of the manuscript.

Author information

Authors and Affiliations



G.F. purified protein, crystallized protein, collected and analyzed data, performed enzyme assays and wrote the paper. R.H.P.L. purified protein, crystallized protein, collected and analyzed data, determined structures, performed enzyme assays and wrote the paper. A.M.B. collected and processed data, determined and analyzed structures and wrote the paper. C.L. cloned GAD67 and produced both recombinant GAD proteins. K.T. performed NMR and enzyme assays. C.J.R. made GAD mutants. N.G.F. assisted with structural analysis. K.M., assisted with kinetic analysis. C.S.H. and J.P.B. provided antibodies and immunological data and analysis. M.W. and J.S. assisted with GAD67 data collection. J.R. assisted with GAD data collection and in writing the paper. O.E.-K. designed the GAD65 expression construct. R.N.P. performed enzyme kinetic analysis. A.I.S. performed mass spectrometry experiments and N-terminal sequencing. I.R.M. analyzed immunological data and provided critical review of the manuscript. M.J.R. co-led the research, analyzed immunological data and provided critical review of the manuscript. J.C.W. led the research, collected and analyzed data, performed structural analysis and wrote the paper.

Corresponding authors

Correspondence to Merrill J Rowley or James C Whisstock.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Fig. 1

Stereo representations of GAD67 active sites. (PDF 1059 kb)

Supplementary Fig. 2

GAD67 catalytic loop electron density. (PDF 679 kb)

Supplementary Fig. 3

GAD67 dimer interface (PDF 2248 kb)

Supplementary Fig. 4

NMR spectroscopic analysis of GABA production (PDF 251 kb)

Supplementary Fig. 5

Molecular surfaces of GAD67 and GAD65 colored according to B-factor. (PDF 1394 kb)

Supplementary Fig. 6

Mutations that affect autoantibody binding to GAD65. (PDF 1088 kb)

Supplementary Fig. 7

Molecular surface of GAD67 colored according to sequence conservation. (PDF 749 kb)

Supplementary Fig. 8

Structural superposition of GAD67 with pig dopa decarboxylase (DDC). (PDF 504 kb)

Supplementary Fig. 9

Proposed reaction mechanism of GAD. (PDF 285 kb)

Supplementary Fig. 10

Initial velocity conditions for GAD assays. (PDF 227 kb)

Supplementary Table 1

Interactions between catalytic loop and rest of protein. (PDF 49 kb)

Supplementary Table 2

Physical and chemical nature of the dimer interfaces. (PDF 28 kb)

Supplementary Table 3

Comparative analysis of sequence differences between GAD65 and GAD67. (PDF 110 kb)

Supplementary Table 4

Initial velocity of GAD variants before and after incubation with 5 mM glutamate. (PDF 58 kb)

Supplementary Table 5

Sequences of primers used for site-directed mutagenesis. (PDF 38 kb)

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Fenalti, G., Law, R., Buckle, A. et al. GABA production by glutamic acid decarboxylase is regulated by a dynamic catalytic loop. Nat Struct Mol Biol 14, 280–286 (2007).

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