Open conformation of human DOPA decarboxylase reveals the mechanism of PLP addition to Group II decarboxylases

Giardina, G.; Montioli, Riccardo; Gianni, Stefano; Cellini, Barbara; Paiardini, Alessandro; Voltattorni, Carla Borri; Cutruzzolà, Francesca

doi:10.1073/pnas.1111456108

Cited by 94 publications

(103 citation statements)

References 41 publications

(46 reference statements)

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“…The structure is nearly identical to the open form of human glutamate decarboxylase (GAD65), with an overall α-carbon RMSD of 1.07Å (Fenalti et al, 2007). Similar comparisons can be made to human aromatic amino acid decarboxylase (Giardina et al, 2011) and wild boar DOPA decarboxylase (Burkhard et al, 2001), with α-carbon RMSDs of 1.21Å and 1.23Å, respectively. The agreement between these structures highlights the commonality of this enzymatic fold, even across multiple species.…”

Section: Resultsmentioning

confidence: 77%

Discovery and Characterization of Gut Microbiota Decarboxylases that Can Produce the Neurotransmitter Tryptamine

Williams

Benschoten

Cimermančič

et al. 2014

Cell Host & Microbe

473

388

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Summary Several recent studies describe the influence of the gut microbiota on host brain and behavior. However, the mechanisms responsible for microbiota-nervous system interactions are unknown. Using a combination of genetics, biochemistry, and crystallography, we identify and characterize two phylogenetically distinct enzymes found in the human microbiome that decarboxylate tryptophan to form the β-arylamine neurotransmitter tryptamine. Although this enzymatic activity is exceedingly rare among bacteria more broadly, analysis of the Human Microbiome Project data demonstrates that at least 10% of the human population harbors at least one bacterium encoding a tryptophan decarboxylase in their gut community. Our results uncover a previously unrecognized enzymatic activity that can give rise to host-modulatory compounds and suggests a potential direct mechanism by which gut microbiota can influence host physiology, including behavior.

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Section: Resultsmentioning

confidence: 77%

Discovery and Characterization of Gut Microbiota Decarboxylases that Can Produce the Neurotransmitter Tryptamine

Williams

Benschoten

Cimermančič

et al. 2014

Cell Host & Microbe

473

388

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“…Although similar conformational changes have been observed for AADC and histidine decarboxylase (4,14), the mechanisms underlying the conformational changes between holo and apo forms of the other members of group II decarboxylases have not been characterized. The crystal structures of both holoGAD65 and holoGAD67 (6) provided an initial opportunity to examine the contrasting autoantigenicities of the GAD isoforms (15,16), which have been mostly correlated to the higher mobility and charge in the C-terminal domain (CTD) of GAD65 (residues 464-585) compared with that of GAD67 (residues 473-594).…”

Section: Significancementioning

confidence: 94%

“…Because they are implicated in a wide range of biological activities from central homeostatic functions to cognitive phenomena, it is not surprising that their activities need to be finely regulated. In this respect, the binding of the cofactor PLP to the apo forms of these enzymes might represent an important mechanism for regulation (4). The interaction of apoGAD with PLP is a major factor in the short-term regulation of GAD activity (5).…”

mentioning

confidence: 99%

Cofactor-dependent conformational heterogeneity of GAD65 and its role in autoimmunity and neurotransmitter homeostasis

Kass

Hoke²,

Costa

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

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The human neuroendocrine enzyme glutamate decarboxylase (GAD) catalyses the synthesis of the inhibitory neurotransmitter gammaaminobutyric acid (GABA) using pyridoxal 5′-phosphate as a cofactor. GAD exists as two isoforms named according to their respective molecular weights: GAD65 and GAD67. Although cytosolic GAD67 is typically saturated with the cofactor (holoGAD67) and constitutively active to produce basal levels of GABA, the membrane-associated GAD65 exists mainly as the inactive apo form. GAD65, but not GAD67, is a prevalent autoantigen, with autoantibodies to GAD65 being detected at high frequency in patients with autoimmune (type 1) diabetes and certain other autoimmune disorders. The significance of GAD65 autoinactivation into the apo form for regulation of neurotransmitter levels and autoantibody reactivity is not understood. We have used computational and experimental approaches to decipher the nature of the holo → apo conversion in GAD65 and thus, its mechanism of autoinactivation. Molecular dynamics simulations of GAD65 reveal coupling between the C-terminal domain, catalytic loop, and pyridoxal 5′-phosphate-binding domain that drives structural rearrangement, dimer opening, and autoinactivation, consistent with limited proteolysis fragmentation patterns. Together with small-angle X-ray scattering and fluorescence spectroscopy data, our findings are consistent with apoGAD65 existing as an ensemble of conformations. Antibody-binding kinetics suggest a mechanism of mutually induced conformational changes, implicating the flexibility of apoGAD65 in its autoantigenicity. Although conformational diversity may provide a mechanism for cofactor-controlled regulation of neurotransmitter biosynthesis, it may also come at a cost of insufficient development of immune self-tolerance that favors the production of GAD65 autoantibodies.conformational dynamics | normal mode analysis | GABA biosynthesis | immunogenicity | autoepitopes T he two mammalian isoforms of glutamate decarboxylase (GAD; EC 4.1.1.15), GAD65 and GAD67, are responsible for the biosynthesis of the inhibitory neurotransmitter gamma-aminobutyric acid (GABA) (1, 2). GAD65 and GAD67 belong to the group II pyridoxal 5′-phosphate (PLP)-dependent enzymes of fold type I, which comprise two other evolutionarily related human enzymes: aromatic L-amino acid decarboxylase (AADC; synonym: L-dopa decarboxylase; EC 4.1.1.28) and histidine decarboxylase (EC 4.1.1.22) (3). Expression of these enzymes in the brain is responsible for the synthesis of the biogenic amines GABA, dopamine, serotonin, and histamine. Because they are implicated in a wide range of biological activities from central homeostatic functions to cognitive phenomena, it is not surprising that their activities need to be finely regulated. In this respect, the binding of the cofactor PLP to the apo forms of these enzymes might represent an important mechanism for regulation (4). The interaction of apoGAD with PLP is a major factor in the short-term regulation of GAD activity (5). Although GAD67 i...

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“…Under this assumption, it may be hypothesized that the HTH domains are able to interact freely with both direct and inverted repeats, even when these are separated by DNA spacers of variable lengths. Many fold-type I PLP-dependent enzymes undergo a large conformational change upon binding of their cofactor PLP [34][35][36]. Changes in protein structure mainly involve loops that form the active site.…”

Section: Apo-and Holo-bcpdxr Have Different Conformationsmentioning

confidence: 99%

Molecular mechanism of PdxR – a transcriptional activator involved in the regulation of vitamin B₆ biosynthesis in the probiotic bacterium Bacillus clausii

et al. 2015

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Pyridoxal 5′‐phosphate (PLP), the well‐known active form of vitamin B6, is an essential enzyme cofactor involved in a large number of metabolic processes. PLP levels need to be finely tuned in response to cell requirements; however, little is known about the regulation of PLP biosynthesis and recycling pathways. The transcriptional regulator PdxR activates transcription of the pdxST genes encoding PLP synthase. It is characterized by an N‐terminal helix‐turn‐helix motif that binds DNA and an effector‐binding C‐terminal domain homologous to PLP‐dependent enzymes. Although it is known that PLP acts as an anti‐activator, the mechanism of action of PdxR is unknown. In the present study, we analyzed the biochemical and DNA‐binding properties of PdxR from the probiotic Bacillus clausii. Spectroscopic measurements showed that PLP is the only B6 vitamer that acts as an effector molecule of PdxR. Binding of PLP to PdxR determines a protein conformational change, as detected by gel filtration chromatography and limited proteolysis experiments. We showed that two direct repeats and one inverted repeat are present in the DNA promoter region and PdxR is able to bind DNA fragments containing any combination of two of them. However, when PLP binds to PdxR, it modifies the DNA‐binding properties of the protein, making it selective for inverted repeats. A molecular mechanism is proposed in which the two different DNA binding modalities of PdxR determined by the presence or absence of PLP are responsible for the control of pdxST transcription.

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Open conformation of human DOPA decarboxylase reveals the mechanism of PLP addition to Group II decarboxylases

Cited by 94 publications

References 41 publications

Discovery and Characterization of Gut Microbiota Decarboxylases that Can Produce the Neurotransmitter Tryptamine

Discovery and Characterization of Gut Microbiota Decarboxylases that Can Produce the Neurotransmitter Tryptamine

Cofactor-dependent conformational heterogeneity of GAD65 and its role in autoimmunity and neurotransmitter homeostasis

Molecular mechanism of PdxR – a transcriptional activator involved in the regulation of vitamin B₆ biosynthesis in the probiotic bacterium Bacillus clausii

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Open conformation of human DOPA decarboxylase reveals the mechanism of PLP addition to Group II decarboxylases

Cited by 94 publications

References 41 publications

Discovery and Characterization of Gut Microbiota Decarboxylases that Can Produce the Neurotransmitter Tryptamine

Discovery and Characterization of Gut Microbiota Decarboxylases that Can Produce the Neurotransmitter Tryptamine

Cofactor-dependent conformational heterogeneity of GAD65 and its role in autoimmunity and neurotransmitter homeostasis

Molecular mechanism of PdxR – a transcriptional activator involved in the regulation of vitamin B6 biosynthesis in the probiotic bacterium Bacillus clausii

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Molecular mechanism of PdxR – a transcriptional activator involved in the regulation of vitamin B₆ biosynthesis in the probiotic bacterium Bacillus clausii