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Human succinic semialdehyde dehydrogenase is a mitochondrial enzyme fundamental in the neurotransmitter γ‐aminobutyric acid catabolism. It catalyzes the NAD+‐dependent oxidative degradation of its derivative, succinic semialdehyde, to succinic acid. Mutations in its gene lead to an inherited neurometabolic rare disease, succinic semialdehyde dehydrogenase deficiency, characterized by mental and developmental delay. Due to the poor characterization of this enzyme, we carried out evolutionary and kinetic investigations to contribute to its functional behavior, a prerequisite to interpreting pathogenic variants. An in silico analysis shows that succinic semialdehyde dehydrogenases belong to two families, one human‐like and the other of bacterial origin, differing in the oligomeric state and in a network of active site residues. This information is coupled to the biophysical–biochemical characterization of the human recombinant enzyme uncovering that (i) catalysis proceeds by an ordered bi–bi mechanism with NAD+ binding before the aldehyde that exerts a partial non‐competitive inhibition; (ii) a stabilizing complex between the catalytic Cys340 and NAD+ is observed and interpreted as a protective mechanism; and (iii) a concerted non‐covalent network assists the action of the catalytic residues Cys340 and Glu306. Through mutational analyses of Lys214, Glu306, Cys340, and Glu515 associated with pH studies, we showed that NAD+ binding is controlled by the dyad Lys214‐Glu515. Moreover, catalysis is assured by proton transfer exerted by the same dyad networked with the catalytic Glu306, involved in catalytic Cys340 deprotonation/reprotonation. The identification of this weak bond network essential for cofactor binding and catalysis represents a first step to tackling the molecular basis for its deficiency. [ABSTRACT FROM AUTHOR]

Succinic semialdehyde dehydrogenase (SSADH) is a mitochondrial enzyme involved in the catabolism of the neurotransmitter γ-amino butyric acid. Pathogenic variants in the gene encoding this enzyme cause SSADH deficiency, a developmental disease that manifests as hypotonia, autism, and epilepsy. SSADH deficiency patients usually have family-specific gene variants. Here, we describe a family exhibiting four different SSADH variants: Val90Ala, Cys93Phe, and His180Tyr/Asn255Asp (a double variant). We provide a structural and functional characterization of these variants and show that Cys93Phe and Asn255Asp are pathogenic variants that affect the stability of the SSADH protein. Due to the impairment of the cofactor NAD+ binding, these variants show a highly reduced enzyme activity. However, Val90Ala and His180Tyr exhibit normal activity and expression. The His180Tyr/Asn255Asp variant exhibits a highly reduced activity as a recombinant species, is inactive, and shows a very low expression in eukaryotic cells. A treatment with substances that support protein folding by either increasing chaperone protein expression or by chemical means did not increase the expression of the pathogenic variants of the SSADH deficiency patient. However, stabilization of the folding of pathogenic SSADH variants by other substances may provide a treatment option for this disease. [ABSTRACT FROM AUTHOR]

Neurodevelopment is a highly organized and complex process involving lasting and often irreversible changes in the central nervous system. Inherited disorders of neurotransmission (IDNT) are a group of genetic disorders where neurotransmission is primarily affected, resulting in abnormal brain development from early life, manifest as neurodevelopmental disorders and other chronic conditions. In principle, IDNT (particularly those of monogenic causes) are amenable to gene replacement therapy via precise genetic correction. However, practical challenges for gene replacement therapy remain major hurdles for its translation from bench to bedside. We discuss key considerations for the development of gene replacement therapies for IDNT. As an example, we describe our ongoing work on gene replacement therapy for succinic semialdehyde dehydrogenase deficiency, a GABA catabolic disorder. [ABSTRACT FROM AUTHOR]

Aim: To elucidate the etiological aspects of autism spectrum disorder (ASD) in succinic semialdehyde dehydrogenase deficiency (SSADHD), related to dysregulation of γ‐aminobutyric acid (GABA) and the imbalance of excitatory and inhibitory neurotransmission. Method: In this prospective, international study, individuals with SSADHD underwent neuropsychological assessments, as well as biochemical, neurophysiological, and neuroimaging evaluations. Results: Of the 29 individuals (17 females) enrolled (median age [IQR] 10 years 5 months [5 years 11 months–18 years 1 month]), 16 were diagnosed with ASD. ASD severity significantly increased with age (r = 0.67, p < 0.001) but was inversely correlated with plasma GABA (r = −0.67, p < 0.001) and γ‐hydroxybutyrate levels (r = −0.538, p = 0.004), and resting motor threshold as measured by transcranial magnetic stimulation (r = −0.44, p = 0.03). A discriminative analysis indicated that an age older than 7 years 2 months (p = 0.004) and plasma GABA levels less than 2.47 μM (p = 0.01) are the threshold values beyond which the likelihood of ASD presenting in individuals with SSADHD is increased. Interpretation: ASD is prevalent but not universal in SSADHD, and it can be predicted by lower levels of plasma GABA and GABA‐related metabolites. ASD severity in SSADHD increases with age and the loss of cortical inhibition. These findings add insight into the pathophysiology of ASD and may facilitate its early diagnosis and intervention in individuals with SSADHD. [ABSTRACT FROM AUTHOR]

Human aromatic amino acid decarboxylase (AADC) is a pyridoxal 5′‐phosphate‐dependent enzyme responsible for the biosynthesis of dopamine and serotonin, essential neurotransmitters involved in motor and cognitive abilities. Mutations in its gene lead to AADC deficiency, a monogenic rare neurometabolic childhood parkinsonism characterized by severe motor and neurodevelopmental symptoms. Here, for the first time, we solved the crystal structure of human holoAADC in the internal aldimine (1.9 Å) and in the external aldimine (2.4 Å) of the substrate analog L‐Dopa methylester. In this intermediate, the highly flexible AADC catalytic loop (CL) is captured in a closed state contacting all protein domains. In addition, each active site, composed by residues of both subunits, is connected to the other through weak interactions and a central cavity. By combining crystallographic analyses with all‐atom and coarse‐grained molecular dynamics simulations, SAXS investigations and limited proteolysis experiments, we realized that the functionally obligate homodimeric AADC enzyme in solution is an elongated, asymmetric molecule, where the fluctuations of the CL are coupled to flexibility at the edge between the N‐terminal and C‐terminal domains. The structural integrity of this peripheral protein region is essential to catalysis, as assessed by both artificial and 37 AADC deficiency pathogenic variants leading to the interpretation that structural dynamics in protein regions far from the active site is essential for CL flexibility and the acquirement of a correct catalytically competent structure. This could represent the molecular basis for pathogenicity prediction in AADC deficiency. [ABSTRACT FROM AUTHOR]

Objective: Succinic semialdehyde dehydrogenase deficiency (SSADHD) is a rare inherited metabolic disorder caused by a defect of γ‐aminobutyrate (GABA) catabolism. Despite the resultant hyper‐GABAergic environment facilitated by the metabolic defect, individuals with this disorder have a paradoxically high prevalence of epilepsy. We aimed to study the characteristics of epilepsy in SSADHD and its concordance with GABA‐related metabolites and neurophysiologic markers of cortical excitation. Methods: Subjects in an international natural history study of SSADHD underwent clinical assessments, electroencephalography, transcranial magnetic stimulation (TMS), magnetic resonance spectroscopy for GABA/N‐acetyl aspartate quantification, and plasma GABA‐related metabolite measurements. Results: A total of 61 subjects with SSADHD and 42 healthy controls were included in the study. Epilepsy was present in 49% of the SSADHD cohort. Over time, there was an increase in severity in 33% of the subjects with seizures. The presence of seizures was associated with increasing age (p =.001) and lower levels of GABA (p =.002), γ‐hydroxybutyrate (GHB; p =.004), and γ‐guanidinobutyrate (GBA; p =.003). Seizure severity was associated with increasing age and lower levels of GABA‐related metabolites as well as lower TMS‐derived resting motor thresholds (p =.04). The cutoff values with the highest discriminative ability to predict seizures were age > 9.2 years (p =.001), GABA < 2.57 μmol·L−1 (p =.002), GHB < 143.6 μmol·L−1 (p =.004), and GBA <.075 μmol·L−1 (p =.007). A prediction model for seizures in SSADHD was comprised of the additive effect of older age and lower plasma GABA, GHB, and GBA (area under the receiver operating characteristic curve of.798, p =.008). Significance: Epilepsy is highly prevalent in SSADHD, and its onset and severity correlate with an age‐related decline in GABA and GABA‐related metabolite levels as well as TMS markers of reduced cortical inhibition. The reduction of GABAergic activity in this otherwise hyper‐GABAergic disorder demonstrates a concordance between epileptogenesis and compensatory responses. These findings may furthermore inform the timing of molecular interventions for SSADHD. [ABSTRACT FROM AUTHOR]

Aromatic l-amino acid decarboxylase (AADC) deficiency is a complex inherited neurological disorder of monoamine synthesis which results in dopamine and serotonin deficiency. The majority of affected individuals have variable, though often severe cognitive and motor delay, with a complex movement disorder and high risk of premature mortality. For most, standard pharmacological treatment provides only limited clinical benefit. Promising gene therapy approaches are emerging, though may not be either suitable or easily accessible for all patients. To characterize the underlying disease pathophysiology and guide precision therapies, we generated a patient-derived midbrain dopaminergic neuronal model of AADC deficiency from induced pluripotent stem cells. The neuronal model recapitulates key disease features, including absent AADC enzyme activity and dysregulated dopamine metabolism. We observed developmental defects affecting synaptic maturation and neuronal electrical properties, which were improved by lentiviral gene therapy. Bioinformatic and biochemical analyses on recombinant AADC predicted that the activity of one variant could be improved by l-3,4-dihydroxyphenylalanine (l-DOPA) administration; this hypothesis was corroborated in the patient-derived neuronal model, where l-DOPA treatment leads to amelioration of dopamine metabolites. Our study has shown that patient-derived disease modelling provides further insight into the neurodevelopmental sequelae of AADC deficiency, as well as a robust platform to investigate and develop personalized therapeutic approaches. [ABSTRACT FROM AUTHOR]

The versatility of reactions catalyzed by pyridoxal 5′-phosphate (PLP) enzymes is largely due to the chemistry of their extraordinary catalyst. PLP is necessary for many reactions involving amino acids. Reaction specificity is controlled by the orientation of the external aldimine intermediate that is formed upon addition of the amino acidic substrate to the coenzyme. The breakage of a specific bond of the external aldimine gives rise to a carbanionic intermediate. From this point, the different reaction pathways diverge leading to multiple activities: transamination, decarboxylation, racemization, elimination, and synthesis. A significant novelty appeared approximately 30 years ago when it was reported that some PLP-dependent decarboxylases are able to consume molecular oxygen transforming an amino acid into a carbonyl compound. These side paracatalytic reactions could be particularly relevant for human health, also considering that some of these enzymes are responsible for the synthesis of important neurotransmitters such as γ-aminobutyric acid, dopamine, and serotonin, whose dysregulation under oxidative conditions could have important implications in neurodegenerative states. However, the reactivity of PLP enzymes with dioxygen is not confined to mammals/animals. In fact, some plant PLP decarboxylases have been reported to catalyze oxidative reactions producing carbonyl compounds. Moreover, other recent reports revealed the existence of new oxidase activities catalyzed by new PLP enzymes, MppP, RohP, Ind4, CcbF, PvdN, Cap15, and CuaB. These PLP enzymes belong to the bacterial and fungal kingdoms and are present in organisms synthesizing bioactive compounds. These new PLP activities are not paracatalytic and could only scratch the surface on a wider and unexpected catalytic capability of PLP enzymes. [ABSTRACT FROM AUTHOR]

Abstract: Aromatic amino acid or Dopa decarboxylase (AADC or DDC) is a homodimeric pyridoxal 5’‐phosphate (PLP) enzyme responsible for the generation of the neurotransmitters dopamine and serotonin. AADC deficiency is a rare inborn disease caused by mutations of the AADC gene leading to a defect of AADC enzyme and resulting in impaired dopamine and serotonin synthesis. Until now, only the molecular effects of homozygous mutations were analyzed. However, although heterozygous carriers of AADC deficiency were identified, the molecular aspects of their enzymatic phenotypes are not yet investigated. Here, we focus our attention on the R347Q/R358H and R347Q/R160W heterozygous mutations, and report for the first time the isolation and characterization, in the purified recombinant form, of the R347Q/R358H heterodimer and of the R358H homodimer. The results, integrated with those already known of the R347Q homodimeric variant, provide evidence that (i) the R358H mutation strongly reduces the PLP‐binding affinity and the catalytic activity, and (ii) a positive interallelic complementation exists between the R347Q and the R358H mutations. Bioinformatics analyses provide the structural basis for these data. Unfortunately, the R347Q/R160W heterodimer was not obtained in a sufficient amount to allow its purification and characterization. Nevertheless, the biochemical features of the R160W homodimer give a contribution to the enzymatic phenotype of the heterozygous R347Q/R160W and suggest the possible relevance of Arg160 in the proper folding of human DDC. © 2018 IUBMB Life, 70(3):215–223, 2018 [ABSTRACT FROM AUTHOR]

These investigations can study muscle modifications and possibly relate them to movement disorders and impairment, a characteristic feature of PD. Mutations in the genes coding for enzymes/proteins related to neurotransmitter synthesis, metabolism, and transport lead to a variety of inherited deficiencies, globally referred to as neurometabolic disorders. Some of these disorders can be treated pharmacologically; in other cases, the current therapies are only palliative, and thus the identification of specific monoamine pathway defects would enable the selection and development of disease-specific new drugs. [Extracted from the article]

Pyridoxal 5′-phosphate (PLP)-dependent enzymes catalyze a wide range of reactions of amino acids and amines, with the exception of glycogen phosphorylase which exhibits peculiar both substrate preference and chemical mechanism. They represent about 4% of the gene products in eukaryotic cells. Although structure-function investigations regarding these enzymes are copious, their regulation by post-translational modifications is largely unknown. Protein phosphorylation is the most common post-translational modification fundamental in mediating diverse cellular functions. This review aims at summarizing the current knowledge on regulation of PLP enzymes by phosphorylation. Starting from the paradigmatic PLP-dependent glycogen phosphorylase, the first phosphoprotein discovered, we collect data in literature regarding functional phosphorylation events of eleven PLP enzymes belonging to different fold types and discuss the impact of the modification in affecting their activity and localization as well as the implications on the pathogenesis of diseases in which many of these enzymes are involved. The pivotal question is to correlate the structural consequences of phosphorylation among PLP enzymes of different folds with the functional modifications exerted in terms of activity or conformational changes or others. Although the literature shows that the phosphorylation of PLP enzymes plays important roles in mediating diverse cellular functions, our recapitulation of clue findings in the field makes clear that there is still much to be learnt. Besides mass spectrometry-based proteomic analyses, further biochemical and structural studies on purified native proteins are imperative to fully understand and predict how phosphorylation regulates PLP enzymes and to find the relationship between addition of a phosphate moiety and physiological response. [ABSTRACT FROM AUTHOR]

Toxoplasma gondii is a protozoan parasite of medical and veterinary relevance responsible for toxoplasmosis in humans. As an efficacious vaccine remains a challenge, chemotherapy is still the most effective way to combat the disease. In search of novel druggable targets, we performed a thorough characterization of the putative pyridoxal 50-phosphate (PLP)-dependent enzyme ornithine aminotransferase from T. gondii ME49 (TgOAT). We overexpressed the protein in Escherichia coli and analysed its molecular and kinetic properties by UV-visible absorbance, fluorescence and CD spectroscopy, in addition to kinetic studies of both the steady state and pre-steady state. TgOAT is largely similar to OATs from other species regarding its general transamination mechanism and spectral properties of PLP; however, it does not show a specific ornithine aminotransferase activity like its human homologue, but exhibits both N-acetylornithine and γ-aminobutyric acid (GABA) transaminase activity in vitro, suggesting a role in both arginine and GABA metabolism in vivo. The presence of Val79 in the active site of TgOAT in place of Tyr, as in its human counterpart, provides the necessary room to accommodate N-acetylornithine and GABA, resembling the active site arrangement of GABA transaminases. Moreover, mutation of Val79 to Tyr results in a change of substrate preference between GABA, N-acetylornithine and L-ornithine, suggesting a key role of Val79 in defining substrate specificity. The findings that TgOAT possesses parasite-specific structural features as well as differing substrate specificity from its human homologue make it an attractive target for anti-toxoplasmosis inhibitor design that can be exploited for chemotherapeutic intervention. [ABSTRACT FROM AUTHOR]