Aug 2026· Food Chemistry· Vol 525 Pt 4, pp.
150639
· 0 citations· 39 references
Medicine
TL;DR
Sulfate is established as a bifunctional modulator with potential applications in the enzymatic production of GABA in the absence of its cofactor pyridoxal-5'-phosphate.
Abstract
Glutamate decarboxylase (GAD) catalyzes the conversion of glutamate to γ-aminobutyric acid (GABA), which is significant for human health and industrial GABA production. This study demonstrates that sulfate acts as an activator for Bacteroides thetaiotaomicron GAD (BTGAD) in the absence of its cofactor pyridoxal-5'-phosphate (PLP), enhancing enzymatic activity by approximately 1.4-fold. However, sulfate competitively inhibits PLP binding and suppresses BTGAD-PLP activity to 36.15%. Structural studies revealed that sulfate binds in a bipartite manner to BTGAD at PLP-binding site and N-terminal sequence. Mutagenesis studies confirm that both binding sites are essential for sulfate-dependent regulation of BTGAD. The mutants H273A and ∆N increased the activation fold of Na2SO4 on BTGAD and enhanced the inhibitory effects of Na2SO4 on BTGAD-PLP. These results establish sulfate as a bifunctional modulator with potential applications in the enzymatic production of GABA.
Despite the exponential growth in genome sequencing, the functional annotation of genes, particularly the discovery of novel enzymatic activities, remains a formidable challenge. This gap is exacerbated by annotation biases that propagate assumptions about enzyme function across homologous sequences. Here, we report the discovery of a previously unrecognized enzymatic activity within a presumed well-characterized enzyme family. The gene rv2531c, under strong purifying selection across Mycobacterium tuberculosis strains, has been annotated as a member of the lysine-ornithine-arginine (KOR) decarboxylase superfamily, which includes over 26,000 sequences. Contrary to this annotation, we show that Rv2531c does not decarboxylate KOR substrates. Instead, using an integrative approach combining bioinformatics, microbiology, metabolomics, and enzymology, we demonstrate that Rv2531c is a novel L-glutamate decarboxylase that sustains carbon flux through the GABA shunt in M. tuberculosis. This newly identified subfamily of enzymes is conserved across Bacteria, Archaea, and Eukarya, and exhibits distinct structural and kinetic features, including an additional domain, hysteresis, and strong positive cooperativity. These characteristics differentiate it from canonical, enterobacterial KOR decarboxylases. More broadly, our findings challenge the narrow substrate and functional scope traditionally assigned to the KOR-DC superfamily. We propose that many members of this large and diverse enzyme family catalyze distinct reactions and participate in previously unrecognized metabolic pathways, revealing a broader and more nuanced role for PLP-dependent enzymes in microbial physiology and evolution.
H. M. Thai, Deborah M. Hunt, Yugen Miyahara et al.· Proceedings of the National...· 0 citations
Many plant species accumulate proline as a mechanism to withstand various abiotic stress conditions. Proline synthesis proceeds from either glutamate or ornithine, but in both pathways the last step is catalysed by a δ1-pyrroline-5-carboxylate (P5C) reductase (EC 1.5.1.12) showing substrate ambiguity. However, the use of NADH or NADPH implies different turnover number and post-translational regulation: physiological levels of free proline feed-back inhibit only the NADH-dependent activity, whereas Cl- anions in the 20 to 200 mM range stimulate the catalytic rate only if NADPH is the hydride donor. To elucidate the molecular basis of such differences we focused on Ser238 of Arabidopsis thaliana P5C reductase, a highly conserved amino acid residue that is involved in P5C binding and had been shown to form a hydrogen bond with aminomethylene-bisphosphonic acids, leading to enzyme inhibition. Site-directed mutagenesis allowed to obtain a mutated form of the enzyme in which Ser238 has been replaced with Ala. The p.Ser238Ala P5C reductase has been expressed in E. coli, affinity purified and thoroughly characterized. Results showed the abolishment of most differences when using either nicotinamide adenine dinucleotide as the cosubstrate. Data represent a first step toward protein engineering aiming at modulating proline biosynthesis under stress.
G. Forlani, Alessandro Martucci· Plant physiology and biochem...· 0 citations
Phosphoribulokinase (PRK) catalyzes the ATP-dependent phosphorylation of ribulose 5-phosphate (Ru5P) to produce ribulose 1,5-bisphosphate in the Calvin-Benson-Bassham cycle. Of PRK's four classifications, class III consists of PRKs from pseudomonadota and α-cyanobacteria. They are known as octameric enzymes regulated allosterically by NADH. In this study, we characterized the PRK from a hydrogen- and sulfur-oxidizing chemolithoautotroph Hydrogenovibrio marinus MH-110 (HmPRK). Phylogenetic analysis revealed that PRKs from organisms of Piscirickettsiaceae, including H. marinus, formed a clearly separate clade within class III. HmPRK lacks most of the conserved arginine residues which are involved in regulation by NADH. Analysis of the PRK activity in the cell-free extract of H. marinus and the purified recombinant HmPRK (rHmPRK) expressed in Escherichia coli revealed the activity as unaffected by NADH. The molecular mass of rHmPRK estimated from size-exclusion chromatography was ca. 75.4 kDa, suggesting it might form a dimer. The optimum temperature and pH of rHmPRK were 30 °C and 7.4, respectively. The enzyme had apparent Km (Ru5P) of 495 ± 108 μM and apparent Km (ATP) of 526 ± 253 μM; apparent Vmax of 298 ± 22 μmol ATP consumed min-1 mg protein-1. Regarding metabolite regulation, the enzyme activity was inhibited by phosphoenolpyruvate and slightly by AMP. These findings indicate that HmPRK has unique properties distinct from those of the canonical class III PRKs.
Tomotaka Jitsukawa, Toya Tamura, Tetsuya Ohtaki et al.· Biochemical and Biophysical...· 0 citations
: Glucose dehydrogenase (GDH) from Bacillus megaterium IWG3 is a NAD(P)⁺ -dependent oxidoreductase widely used in biosensing and biocatalytic NADPH regeneration. However, its intrinsic preference for NAD⁺ over NADP⁺ limits its application in NADPH -driven processes. Here, we report a structure-guided rational design to invert the coenzyme specificity of GDH by targeting a single residue within the conserved GXXXGXG motif of the Rossmann fold. Molecular docking and structural analysis identified Thr17 as the key residue forming a hydrogen bond with the 2′ - hydroxyl of NAD⁺, thereby discriminating against the 2′ -phosphate of NADP⁺. Three -point mutants—T17G, T17K, and T17R—were constructed, expressed, and kinetically characterized. The T17G mutation dramatically inverted cofactor preference, increasing the catalytic efficiency ratio (NADP⁺/NAD⁺) from 0.78 (wild -type) to 7.5, driven by a 2.4-fold decrease for NADP⁺ and a 4.6 - fold increase in for NAD⁺. Remarkably, the T17K mutant not only shifted preference toward NADP⁺ (specificity ratio 0.96) but also enhanced turnover numbers for both coenzymes by up to 5.2-fold, achieving c atalytic efficiencies of 6.39 mM⁻¹·s⁻¹ (NAD⁺) and 6.15 mM⁻¹·s⁻¹ (NADP⁺) —the highest among all variants tested. In contrast, the T17R mutation severely impaired NADP⁺ binding ( k m = 97.18 mM) and abolished activity. Structural modeling revealed that glycine c reates space to accommodate the 2′ -phosphate, while lysine establishes a favorable electrostatic interaction with the phosphate group; arginine’s bulky guanidinium group causes steric clash. This study demonstrates that a single, rationally designed mutation at position 17 can simultaneously broaden cofactor specificity and improve catalytic efficiency, with the T17K mutant emerging as a superior biocatalyst for NADPH regeneration. The strategy provides a generalizable framework for engineering cofactor preference in short-chain dehydrogenase/reductase family enzymes.
Y. Shen, Ke-Ju Jing· International Journal of Fro...· 0 citations
Metal ions are essential in biological systems, serving structural and signaling functions, and acting as enzyme cofactors. The d-block transition metals are most commonly found in metalloenzymes, however, recent discoveries have highlighted the biological relevance of the rare earth elements (REEs). Despite growing evidence of lanthanide utilization in biology, our understanding of REE handling and function, especially beyond pyrroloquinoline quinone (PQQ)-dependent enzymes in methanotrophic systems, remains limited. In this work, we investigated and biochemically characterized the REE-binding properties of alcohol dehydrogenase D (AdhD), a thermostable aldo-keto reductase (AKR) from Pyrococcus furiosus. We identified and characterized a high-affinity REE binding site with low micromolar affinity, consistent with bioavailable free REE concentrations in the Pyrococcus furiosus natural niche, near geothermal marine sediments. Taking advantage of the REE binding site, we developed an AdhD-based, citrate-assisted REE separation protocol. This purification strategy provides an efficient, single-stage purification platform with high resolution to separate REEs from common divalent contaminants, and it can resolve heavy REEs from each other as an alternative to traditional solvent extraction-based purifications. This strategy offers an alternative to conventional, environmentally detrimental separation methods. These findings expand the understanding of REE binding in noncanonical systems, and showcases AdhD as a robust, thermostable scaffold for REE separations.
S. Abeyrathna, Farid F Khoury, Katarzyna H. Kucharzyk et al.· Journal of Molecular Biology· 0 citations
Findings suggest that the studied FPMO may play a role in antibiotic resistance in P. aeruginosa by oxidatively inactivating ampicillin by oxidatively inactivating ampicillin.
Maliheh Mohammadkhani, Shamsozoha Abolmaali, S. D. Astaneh· Iranian Journal of Microbiol...· 0 citations
