?-Aminobutyric acid (GABA) is a four carbon non-proteinogenic amino acid found in species of all kingdoms. In insects and mammals, GABA acts as an inhibitory neurotransmitter sensed by receptors. In bacteria, fungi and plants, organisms lacking a central nervous system, GABA receptors have not been described yet. However, known GABA-metabolism enzymes are congruent in all species analyzed so far. GABA is produced from glutamate by glutamate decarboxylase (GAD), and catabolism is conducted by GABA transaminase (GABA-T) and succinic semialdehyde dehydrogenase (SSADH) to yield succinate. Alternatively, the intermediate succinic semialdehyde (SSA) can be reduced to ?-hydroxybutyric acid (GHB) instead of being oxidized to succinate; the product GHB has been described as a neuroactive substance in insects and mammals also sensed by receptors. Regulatory enzymes of GABA metabolism have not been described yet.

In human and mouse, the inherited disease GHB aciduria has been attributed to mutations in the SSADH gene, i.e., to a mal- or dysfunction of the SSADH enzyme. Affected mice and patients accumulate GABA and GHB in their brains leading to general neurological impairment, seizures, ataxia and early death. The most common therapy for patients is the administration of vigabatrine (VGB), a GABA analogue inhibiting GABA-T (also in plants). However, patients do not respond uniformly positive to this drug. Several experimental drugs are currently being analyzed in mouse that might lead to the development of therapeutics in the future.

The similarity of the GABA metabolic pathway in mammals and plants and the absence of receptors in the latter predestine the model plant Arabidopsis thaliana as a suitable system to study GABA metabolism. Perturbations in GABA metabolism – in mammals often causing strong (negative) phenotypes due to receptor read out – can be studied in plants without the interference of this direct read out system. We made use of the absence of receptors in our model organism by analyzing suppressors (induced by EMS mutagenesis) of ssadh mutants. We expect to obtain results which might lead to conclusions helping to tackle human GABA-related disorders, e.g. by identifying new target proteins for drug action.

The approach makes use of a collection of EMS-induced suppressors of ssadh mutants. Suppressor mutants (partially) rescue the phenotype of ssadh mutants which is characterized by stunted bushy growth and the development of necrotic leaf lesions caused by accumulation of reactive oxygen species (ROS) most likely elicited by the accumulation of the reactive carbonyl SSA. We are currently sequencing the genomes of four of the suppressor mutants that contain different increased levels of GABA and GHB, compared to wild type, using next generation sequencing. Detection of several mutations caused by EMS treatment together with coarse mapping should lead to the identification of the respective mutation suppressing the ssadh phenotype of each line. Direction of further analyses strongly depends on the gene functions that are impaired in suppressor mutants.

In a second approach, GABA-T will be comparatively analyzed with ?-ornithine aminotransferase (d-OAT). We suspect d-OAT to be a ‘hidden’ GABA-T, i.e., to have a so far undetected affinity for GABA.

Whereas an additional knock out of the GABA-T gene rescues the ssadh phenotype under long-day conditions, short-day growth evokes a phenotype somehow resembling the ssadh phenotype, especially since ROS accumulate and necroses appear. Strikingly, gaba-t single mutants do not display any aberrant phenotype. This finding led us to the assumption that an alternative aminotransferase can form SSA from GABA when its concentration is sufficient to meet the presumably poor k_M of the enzyme. In gaba-t ssadh double mutants GABA content is increased by a factor of about 50, compared to wild type. An additional factor of three is found when plants were grown under short- vs. long-day conditions. The resulting factor of about 150 might be sufficient for GABA to be bound to and converted by the d-OAT.

D-OAT is a good candidate for being the proposed poor GABA-T for several reasons. Like GABA-T (i) the enzyme is located in mitochondria and (ii) it transfers a terminal amino group of its donor. Moreover, (iii) GABA is structurally similar to ?-ornithine, (iv) both enzymes are inhibited by the structural analog gabaculine, and (v) an exchange of a single amino acid of the active site led to a 1000-fold decrease and 16-fold increase of activity with ?-ornithine and GABA as substrate, respectively.

We will analyze enzyme activities of native and mutated d-OATs and GABA-Ts of Arabidopsis and tobacco plants the latter of which as per publication contain a GABA-T activity that uses ?-ketoglutarate (which is also co-substrate of d-OATs) instead of pyruvate. Moreover, a large collection of Arabidopsis mutants exists, among them the gaba-t and d-oat mutants, which will be analyzed and crossed, and can be used for the characterization of the several different native and mutated aminotransferases since they should be free of background activities.

Apart from resolving the described short-day phenotype and from getting insight into structure-function relations of aminotransferases the expected results may help to broaden our knowledge of GABA metabolism in general, i.e., not only in Arabidopsis but also in mammals.

Mutants of GABA transaminase (POP2) suppress the severe phenotype of succinic semialdehyde dehydrogenase (ssadh) mutants in Arabidopsis. Ludewig F, Hüser A, Fromm H, Beauclair L, Bouché N. PLoS One. 2008;3(10)

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