Abstract 1873 The Synthesis of Neuromodulatory Molecules by A Gut Microbial Glutamate Decarboxylase: GABA, Taurine and its Analogs, and β-Alanine

Dysbiosis in the human gut microbiome is associated as a factor contributing to various neurological disorders. However, the mechanism of disease onset due to dysbiosis still remains unclear. Prior research has demonstrated the role of gut microbes in γ-aminobutyric acid (GABA) production, a neurotransmitter pivotal in neurodegenerative diseases, particularly Alzheimer's (AD) and dementia, where consistently lower GABA levels have been reported. Multiple investigations on the microbiomes of individuals suffering from AD and dementia have consistently shown differences in the abundance of microbes from the genus Bacteroides, which encode the gene for the enzyme glutamate decarboxylase (GAD). In this study, we characterized a GAD from a prominent gut microbe, Bacteroides fragilis (BfGAD). We show that the enzyme is able to decarboxylate the substrate L-glutamate (L-Glu) to produce GABA at an acidic pH. The steady-state kinetics studies with BfGAD yield the Km of 9.2±0.7 mM for L-glutamate. Additionally, we explored the possible role of BfGAD in the synthesis of taurine and its derivatives. Taurine, a sulfur-containing amino acid, acts as a potent cytoprotectant and exhibits GABAA receptor agonist properties with downstream effects similar to GABA. Lower taurine levels have been associated with cognitive deficits, whereas taurine supplementation reverses cognitive impairment and enhance learning and memory in murine models. With the help of headspace gas chromatography, thin-layer chromatography (TLC), and liquid chromatography-mass spectrometry (LC-MS), we demonstrate that BfGAD is able to synthesize hypotaurine, taurine, and homotaurine from the decarboxylation of sulfur-containing amino acids L-cysteine sulfinic acid (L-CSA), L-cysteic acid (L-CA), and L-homocysteic acid (L-HCA). In addition, we engineered BfGAD to enhance its efficiency in producing taurine, with a single amino acid modification in a predicted substrate binding pocket. The alteration of Asp104 (D104) residue to asparagine (D104N) shows a 2-fold increase in taurine production. Our study also reveals BfGAD's ability to catalyze the conversion of L-Aspartate (L-ASP) to β-alanine, an additional neuromodulatory molecule. Directed evolution via rational design of BfGAD has provided us with a spectrum of engineered enzymes that either exhibit stringent substrate specificity towards L-glutamate (F81W), have altered substrate specificity for other tested substrates (D104E), or show increased catalytic tolerance at higher pH (T80S). In summary, our work sheds light on the potential for fine-tuning gut microbial glutamate decarboxylase activity for the possible modulation of multiple neuromodulatory molecules. We thank the New College of Interdisciplinary Arts and Sciences (NCIAS) at Arizona State University and the Edson Initiative for Dementia Care and Solutions for their funding support.