Expression analysis of selected genes involved in tryptophan metabolic pathways in Egyptian children with Autism Spectrum Disorder and learning disabilities.

Autism Spectrum Disorder (ASD) and learning disabilities are neurodevelopmental disabilities characterized by dramatically increasing incidence rates, yet the exact etiology for these disabilities is not identified. Impairment in tryptophan metabolism has been suggested to participate in the pathogenesis of ASD, however, further validation of its involvement is required. Additionally, its role in learning disabilities is still uninvestigated. Our objective was to evaluate some aspects of tryptophan metabolism in ASD children (N = 45) compared to children with learning disabilities (N = 44) and healthy controls (N = 40) by measuring the expression levels of the MAOA, HAAO and AADAT genes using real-time RT-qPCR. We also aimed to correlate the expression patterns of these genes with parental ages at the time of childbirth, levels of serum iron, and vitamin D3 and zinc/copper ratio, as possible risk factors for ASD. Results demonstrated a significant decrease in the expression of the selected genes within ASD children (p < 0.001) relative to children with learning disabilities and healthy controls, which significantly associated with the levels of our targeted risk factors (p < 0.05) and negatively correlated to ASD scoring (p < 0.001). In conclusion, this study suggests that the expression of the MAOA, HAAO and AADAT genes may underpin the pathophysiology of ASD.

Genome-wide analyses identify a role for SLC17A4 and AADAT in thyroid hormone regulation.

Thyroid dysfunction is an important public health problem, which affects 10% of the general population and increases the risk of cardiovascular morbidity and mortality. Many aspects of thyroid hormone regulation have only partly been elucidated, including its transport, metabolism, and genetic determinants. Here we report a large meta-analysis of genome-wide association studies for thyroid function and dysfunction, testing 8 million genetic variants in up to 72,167 individuals. One-hundred-and-nine independent genetic variants are associated with these traits. A genetic risk score, calculated to assess their combined effects on clinical end points, shows significant associations with increased risk of both overt (Graves' disease) and subclinical thyroid disease, as well as clinical complications. By functional follow-up on selected signals, we identify a novel thyroid hormone transporter (SLC17A4) and a metabolizing enzyme (AADAT). Together, these results provide new knowledge about thyroid hormone physiology and disease, opening new possibilities for therapeutic targets.

Both BAT1 and ARO8 are responsible for unpleasant odor generation in halo-tolerant yeast Zygosaccharomyces rouxii.

Soy sauce yeast Zygosaccharomyces rouxii plays a central role in the production of flavor compounds in soy sauce, while the flor-forming strain spoils its quality by producing 2-methylpropanoic acid, 2-methylbutanoic acid, and 3-methylbutanoic acid, which have an unpleasant odor. To investigate the relationship between flor formation and unpleasant odor, we measured the volatile compounds that accumulated under various growth conditions. As a result, marked amounts of 2-methylpropanoic acid, 2-methylbutanoic acid, or 3-methylbutanoic acid accumulated in synthetic medium containing valine, isoleucine, or leucine, respectively, under aerobic growth conditions. These results implied that the unpleasant compounds were produced from their corresponding branched chain amino acid (BCAA) when the cell was placed under aerobic condition through flor formation. The first step in BCAA catabolism and the last step in BCAA anabolism are both catalyzed by a BCAA transaminase. A mutant lacking the BCAA transaminase gene, BAT1, resulted in valine and isoleucine auxotrophy, while a mutant lacking both BAT1 and the α-aminoadipate aminotransferase gene, ARO8, resulted in valine, isoleucine, and leucine auxotrophy. Although the bat1∆ aro8∆ double mutant formed flor similarly to the wild-type strain, the mutant exhibited less unpleasant odor generation. These results suggest that the interconversion between 4-methyl-2-oxopentanoate and leucine is catalyzed by both Bat1p and Aro8p in Z. rouxii. Taken together, these results indicate that flor formation is not seemed to be directly linked to unpleasant odor generation. These findings encourage us to breed flor-forming yeasts without an unpleasant odor.

Mechanism of the aromatic aminotransferase encoded by the Aro8 gene from Saccharomyces cerevisiae.

The amino acid L-lysine is synthesized in Saccharomyces cerevisiae via the α-aminoadipate pathway. An as yet unidentified PLP-containing aminotransferase is thought to catalyze the formation of α-aminoadipate from α-ketoadipate in the L-lysine biosynthetic pathway that could be the yeast Aro8 gene product. A screen of several different amino acids and keto-acids showed that the enzyme uses L-tyrosine, L-phenylalanine, α-ketoadipate, and L-α-aminoadipate as substrates. The UV-visible spectrum of the aminotransferase exhibits maxima at 280 and 343 nm at pH 7.5. As the pH is decreased the peak at 343 nm (the unprotonated internal aldimine) disappears and two new peaks at 328 and 400 nm are observed representing the enolimine and ketoenamine tautomers of the protonated aldimine, respectively. Addition, at pH 7.1, of α-ketoadipate to free enzyme leads to disappearance of the absorbance at 343 nm and appearance of peaks at 328 and 424 nm. The V/E(t) and V/K(α-ketoadipate)E(t) pH profiles are pH independent from pH 6.5 to 9.6, while the V/K(L-tyrosine) pH-rate profile decreases below a single pK(a) of 7.0 ± 0.1. Data suggest the active enzyme form is with the internal aldimine unprotonated. We conclude the enzyme should be categorized as a α-aminoadipate aminotransferase.

Dual roles of a conserved pair, Arg23 and Ser20, in recognition of multiple substrates in alpha-aminoadipate aminotransferase from Thermus thermophilus.

To clarify the mechanism for substrate recognition of alpha-aminoadipate aminotransferase (AAA-AT) from Thermus thermophilus, the crystal structure of AAA-AT complexed with N-(5'-phosphopyridoxyl)-l-glutamate (PPE) was determined at 1.67 A resolution. The crystal structure revealed that PPE is recognized by amino acid residues the same as those seen in N-(5'-phosphopyridoxyl)-l-alpha-aminoadipate (PPA) recognition; however, to bind the gamma-carboxyl group of Glu at a fixed position, the Calpha atom of the Glu moiety moves 0.80 A toward the gamma-carboxyl group in the PPE complex. Markedly decreased activity for Asp can be explained by the shortness of the aspartyl side chain to be recognized by Arg23 and further dislocation of the Calpha atom of bound Asp. Site-directed mutagenesis revealed that Arg23 has dual functions for reaction, (i) recognition of gamma (delta)-carboxyl group of Glu (AAA) and (ii) rearrangement of alpha2 helix by changing the interacting partners to place the hydrophobic substrate at the suitable position.

Mechanism for multiple-substrates recognition of alpha-aminoadipate aminotransferase from Thermus thermophilus.

Alpha-aminoadipate aminotransferase (AAA-AT), a homolog of mammalian kynurenine aminotransferase II (Kat II), transfers an amino group to 2-oxoadipate to yield alpha-aminoadipate in lysine biosynthesis through the alpha-aminoadipate pathway in Thermus thermophilus. AAA-AT catalyzes transamination against various substrates, including AAA, glutamate, leucine, and aromatic amino acids. To elucidate the structural change for recognition of various substrates, we determined crystal structures of AAA-AT in four forms: with pyridoxal 5'-phosphate (PLP) (PLP complex), with PLP and leucine (PLP/Leu complex), with N-phosphopyridoxyl-leucine (PPL) (PPL complex), and with N-phosphopyridoxyl-alpha-aminoadipate (PPA) at 2.67, 2.26, 1.75, and 1.67 A resolution, respectively. The PLP complex is in an open state, whereas PLP/Leu, PPL, and PPA complexes are in closed states with maximal displacement (over 7 A) of the alpha2 helix and the beta1 strand in the small domain to cover the active site, indicating that conformational change is induced by substrate binding. In PPL and PLP/Leu complexes, several hydrophobic residues on the alpha2 helix recognize the hydrophobic side chain of the bound leucine moiety whereas, in the PPA complex, the alpha2 helix rotates to place the guanidium moiety of Arg23 on the helix at the appropriate position to interact with the carboxyl side chain of the AAA moiety. These results indicate that AAA-AT can recognize various kinds of substrates using the mobile alpha2 helix. The crystal structures and site-directed mutagenesis revealed that intersubunit-electrostatic interactions contribute to the elevated thermostability of this enzyme.