Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

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

Abstract

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.

This is a preview of subscription content, access via your institution

Relevant articles

Open Access articles citing this article.

Access options

Buy article

Get time limited or full article access on ReadCube.

$32.00

All prices are NET prices.

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.

Accession codes

Primary accessions

Protein Data Bank

Referenced accessions

Protein Data Bank

References

  1. Capitani, G. et al. Crystal structure and functional analysis of Escherichia coli glutamate decarboxylase. EMBO J. 22, 4027–4037 (2003).

    Article  CAS  Google Scholar 

  2. Gut, H. et al. Escherichia coli acid resistance: pH-sensing, activation by chloride and autoinhibition in GadB. EMBO J. 25, 2643–2651 (2006).

    Article  CAS  Google Scholar 

  3. Soghomonian, J.J. & Martin, D.L. Two isoforms of glutamate decarboxylase: why? Trends Pharmacol. Sci. 19, 500–505 (1998).

    Article  CAS  Google Scholar 

  4. Fagiolini, M. et al. Specific GABAA circuits for visual cortical plasticity. Science 303, 1681–1683 (2004).

    Article  CAS  Google Scholar 

  5. Ge, S. et al. GABA regulates synaptic integration of newly generated neurons in the adult brain. Nature 439, 589–593 (2006).

    Article  CAS  Google Scholar 

  6. Nakatsu, Y. et al. A cluster of three GABAA receptor subunit genes is deleted in a neurological mutant of the mouse p locus. Nature 364, 448–450 (1993).

    Article  CAS  Google Scholar 

  7. Asada, H. et al. Cleft palate and decreased brain gamma-aminobutyric acid in mice lacking the 67-kDa isoform of glutamic acid decarboxylase. Proc. Natl. Acad. Sci. USA 94, 6496–6499 (1997).

    Article  CAS  Google Scholar 

  8. Kash, S.F. et al. Epilepsy in mice deficient in the 65-kDa isoform of glutamic acid decarboxylase. Proc. Natl. Acad. Sci. USA 94, 14060–14065 (1997).

    Article  CAS  Google Scholar 

  9. Asada, H. et al. Mice lacking the 65 kDa isoform of glutamic acid decarboxylase (GAD65) maintain normal levels of GAD67 and GABA in their brains but are susceptible to seizures. Biochem. Biophys. Res. Commun. 229, 891–895 (1996).

    Article  CAS  Google Scholar 

  10. Lynex, C.N. et al. Homozygosity for a missense mutation in the 67 kDa isoform of glutamate decarboxylase in a family with autosomal recessive spastic cerebral palsy: parallels with Stiff-Person Syndrome and other movement disorders. BMC Neurol. 4, 20 (2004).

    Article  Google Scholar 

  11. Jansonius, J.N. Structure, evolution and action of vitamin B6-dependent enzymes. Curr. Opin. Struct. Biol. 8, 759–769 (1998).

    Article  CAS  Google Scholar 

  12. Percudani, R. & Peracchi, A. A genomic overview of pyridoxal-phosphate-dependent enzymes. EMBO Rep. 4, 850–854 (2003).

    Article  CAS  Google Scholar 

  13. Porter, T.G., Spink, D.C., Martin, S.B. & Martin, D.L. Transaminations catalysed by brain glutamate decarboxylase. Biochem. J. 231, 705–712 (1985).

    Article  CAS  Google Scholar 

  14. Battaglioli, G., Liu, H. & Martin, D.L. Kinetic differences between the isoforms of glutamate decarboxylase: implications for the regulation of GABA synthesis. J. Neurochem. 86, 879–887 (2003).

    Article  CAS  Google Scholar 

  15. Lernmark, A. Glutamic acid decarboxylase–gene to antigen to disease. J. Intern. Med. 240, 259–277 (1996).

    Article  CAS  Google Scholar 

  16. Battaglioli, G., Liu, H., Hauer, C.R. & Martin, D.L. Glutamate decarboxylase: loss of N-terminal segment does not affect homodimerization and determination of the oxidation state of cysteine residues. Neurochem. Res. 30, 989–1001 (2005).

    Article  CAS  Google Scholar 

  17. Martin, D.L., Liu, H., Martin, S.B. & Wu, S.J. Structural features and regulatory properties of the brain glutamate decarboxylases. Neurochem. Int. 37, 111–119 (2000).

    Article  CAS  Google Scholar 

  18. Law, R.H., Rowley, M.J., Mackay, I.R. & Corner, B. Expression in Saccharomyces cerevisiae of antigenically and enzymatically active recombinant glutamic acid decarboxylase. J. Biotechnol. 61, 57–68 (1998).

    Article  CAS  Google Scholar 

  19. Qu, K., Martin, D.L. & Lawrence, C.E. Motifs and structural fold of the cofactor binding site of human glutamate decarboxylase. Protein Sci. 7, 1092–1105 (1998).

    Article  CAS  Google Scholar 

  20. Kash, S.F., Tecott, L.H., Hodge, C. & Baekkeskov, S. Increased anxiety and altered responses to anxiolytics in mice deficient in the 65-kDa isoform of glutamic acid decarboxylase. Proc. Natl. Acad. Sci. USA 96, 1698–1703 (1999).

    Article  CAS  Google Scholar 

  21. Baekkeskov, S. et al. Identification of the 64K autoantigen in insulin-dependent diabetes as the GABA-synthesizing enzyme glutamic acid decarboxylase. Nature 347, 151–156 (1990).

    Article  CAS  Google Scholar 

  22. Pearce, D.A., Atkinson, M. & Tagle, D.A. Glutamic acid decarboxylase autoimmunity in Batten disease and other disorders. Neurology 63, 2001–2005 (2004).

    Article  CAS  Google Scholar 

  23. Raju, R. et al. Analysis of GAD65 autoantibodies in Stiff-Person Syndrome patients. J. Immunol. 175, 7755–7762 (2005).

    Article  CAS  Google Scholar 

  24. Burkhard, P., Dominici, P., Borri-Voltattorni, C., Jansonius, J.N. & Malashkevich, V.N. Structural insight into Parkinson's disease treatment from drug-inhibited DOPA decarboxylase. Nat. Struct. Biol. 8, 963–967 (2001).

    Article  CAS  Google Scholar 

  25. Bertoldi, M., Gonsalvi, M., Contestabile, R. & Voltattorni, C.B. Mutation of tyrosine 332 to phenylalanine converts dopa decarboxylase into a decarboxylation-dependent oxidative deaminase. J. Biol. Chem. 277, 36357–36362 (2002).

    Article  CAS  Google Scholar 

  26. Eliot, A.C. & Kirsch, J.F. Pyridoxal phosphate enzymes: mechanistic, structural, and evolutionary considerations. Annu. Rev. Biochem. 73, 383–415 (2004).

    Article  CAS  Google Scholar 

  27. Papakonstantinou, T., Law, R.H., Gardiner, P., Rowley, M.J. & Mackay, I.R. Comparative expression and purification of human glutamic acid decarboxylase from Saccharomyces cerevisiae and Pichia pastoris. Enzyme Microb. Technol. 26, 645–652 (2000).

    Article  CAS  Google Scholar 

  28. Evans, P. Scaling and assessment of data quality. Acta Crystallogr. D Biol. Crystallogr. 62, 72–82 (2006).

    Article  Google Scholar 

  29. Leslie, A.G.W. Joint CCP4 + ESF-EAMCB Newsletter on Protein Crystallography No. 26 (1992).

    Google Scholar 

  30. Potterton, E., Briggs, P., Turkenburg, M. & Dodson, E. A graphical user interface to the CCP4 program suite. Acta Crystallogr. D Biol. Crystallogr. 59, 1131–1137 (2003).

    Article  Google Scholar 

  31. Collaborative Computational Project, Number 4. The CCP4 suite: programs for protein crystallography. Acta Crystallogr. D Biol. Crystallogr. 50, 760–763 (1994).

  32. McCoy, A.J., Storoni, L.C. & Read, R.J. Simple algorithm for a maximum-likelihood SAD function. Acta Crystallogr. D Biol. Crystallogr. 60, 1220–1228 (2004).

    Article  Google Scholar 

  33. Jaroszewski, L., Rychlewski, L., Li, Z., Li, W. & Godzik, A. FFAS03: a server for profile–profile sequence alignments. Nucleic Acids Res. 33, W284–W288 (2005).

    Article  CAS  Google Scholar 

  34. Canutescu, A.A., Shelenkov, A.A. & Dunbrack, R.L., Jr. A graph-theory algorithm for rapid protein side-chain prediction. Protein Sci. 12, 2001–2014 (2003).

    Article  CAS  Google Scholar 

  35. Murshudov, G.N., Vagin, A.A. & Dodson, E.J. Refinement of macromolecular structures by the maximum-likelihood method. Acta Crystallogr. D Biol. Crystallogr. 53, 240–255 (1997).

    Article  CAS  Google Scholar 

  36. Emsley, P. & Cowtan, K. Coot: model-building tools for molecular graphics. Acta Crystallogr. D Biol. Crystallogr. 60, 2126–2132 (2004).

    Article  Google Scholar 

  37. Morris, R.J., Perrakis, A. & Lamzin, V.S. ARP/wARP and automatic interpretation of protein electron density maps. Methods Enzymol. 374, 229–244 (2003).

    Article  CAS  Google Scholar 

  38. DeLano, W.L. The PyMOL User's Manual (DeLano Scientific, San Carlos, California, USA, 2002).

    Google Scholar 

  39. Konagurthu, A.S., Whisstock, J.C., Stuckey, P.J. & Lesk, A.M. MUSTANG: a multiple structural alignment algorithm. Proteins 64, 559–574 (2006).

    Article  CAS  Google Scholar 

  40. Martin, S.B. & Martin, D.L. Stimulation by phosphate on the activation of glutamate apodecarboxylase by pyridoxyl-5′-phosphate and its implications for the control of GABA synthesis. J. Neurochem. 33, 1275–1283 (1979).

    Article  CAS  Google Scholar 

  41. Barton, G.J. ALSCRIPT: a tool to format multiple sequence alignments. Protein Eng. 6, 37–40 (1993).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

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

Authors

Contributions

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.

Ethics declarations

Competing interests

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)

Rights and permissions

Reprints and Permissions

About this article

Cite this article

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). https://doi.org/10.1038/nsmb1228

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nsmb1228

This article is cited by

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing