Search Results (187)

Gamma (γ)-aminobutyric acid (GABA) is a non-proteinogenic amino acid recognized for its primary function as the main inhibitory neurotransmitter in the mammalian central nervous system and its role as a stress-responsive metabolite in various microorganisms. This review critically examines the biosynthesis of GABA by lactic acid bacteria (LAB) via the glutamate decarboxylase (GAD) system and evaluates its potential applications in food and health sectors. The mechanistic details of the GAD pathway are analyzed, focusing on the integrated roles of gadA/gadB decarboxylases, the gadC antiporter, and pyridoxal−5′-phosphate (PLP) dependency in relation to acid resistance, metabolic flux, and strain variability. Taxonomic and strain-level diversity among GABA-producing LAB is assessed, with emphasis on the highly strain-specific nature of GABA production rather than broad species or genus generalizations. Fermentation optimization parameters (pH, temperature, substrate loading, and cofactor management) and scale-up challenges, including techno-economic feasibility and downstream recovery efficiency, are critically evaluated. Integration of LAB-derived GABA into fermented food matrices is discussed with attention to sensory compromises, stability, regulatory factors, and clean-label considerations. Evidence from clinical and preclinical studies is synthesized to assess the physiological significance of dietary GABA, distinguishing between purified GABA supplementation, GABA-enriched fermented foods, and probiotic effects of live LAB, while addressing the GABA paradox and gut–brain axis interactions. Significant research gaps are identified, including the need for standardized quantification methodologies, multi-omics-guided strain engineering, predictive bioprocess modeling, and rigorously designed human trials in realistic food matrices. This review provides a systems-oriented, critical framework to promote scalable and evidence-based advancement of GABA-enriched functional foods. Full article

N-acetylornithine aminotransferase (AcOAT) is a pyridoxal 5′-phosphate (PLP)-dependent enzyme that catalyzes a key transamination step in arginine biosynthesis. In cyanobacteria, arginine metabolism is closely associated with nitrogen assimilation and storage, yet biochemical information on cyanobacterial AcOATs remains limited. In this study, the AcOAT encoded by the cce_3094 gene from Crocosphaera subtropica ATCC 51142 (CsAcOAT) was cloned, heterologously expressed, purified, and systematically characterized. Recombinant CsAcOAT was obtained as a soluble protein with an apparent molecular mass of approximately 43 kDa. Steady-state kinetic analysis showed that CsAcOAT catalyzed transamination between N-acetylornithine (AcOrn) and α-ketoglutarate (α-KG), with apparent K_M values of 0.17 ± 0.03 mM for AcOrn and 0.020 ± 0.003 mM for α-KG, indicating a higher affinity for α-KG. The enzyme exhibited optimal activity at pH 8.5 and 30 °C, retained relatively high activity over a broad temperature range of 0–50 °C, and was activated by Zn²⁺ and Co²⁺ but inhibited by Ni²⁺. Structural analysis based on homology modeling, molecular docking, and molecular dynamics simulations suggested a conserved PLP-dependent aminotransferase fold and a stable binding mode for the PLP-AcOrn complex in the active-site pocket. Site-directed mutagenesis further demonstrated that Gly114, Asp239, Lys268, and Thr296 are indispensable for catalytic activity, whereas Ser113, Ala115, and Gln242 make important contributions to catalytic turnover and cofactor-assisted catalysis. These results provide biochemical and structural characterization of CsAcOAT, expand current knowledge of cyanobacterial AcOATs, and offer a useful basis for future studies on arginine metabolism and nitrogen storage in diazotrophic cyanobacteria. Full article

Pyridoxal 5′-phosphate (PLP), the catalytically active form of vitamin B6, is an enabling cofactor for synthetic biocatalysis, but PLP production remains difficult to compare across chemical, microbial, and cell-free routes. This review reframes PLP synthesis as a biocatalytic cascade-design problem. Chemical phosphorylation is used as a benchmark for selectivity, reagent burden, and purification, whereas de novo and salvage-pathway enzymes define the molecular constraints governing biological production. We focus on PdxK/PdxY, PdxH/PNPOx, PdxS/PdxT, engineered acid phosphatase, and polyphosphate kinase modules. Recent PPi-driven and PPK/polyP-supported cascades show that high PLP concentrations are attainable, but titer alone is not sufficient for route comparison: substrate identity, phosphate-donor equivalents, adenylate loading, whole-cell or cell-free format, oxygen and H₂O₂ management, catalyst reuse, salt burden, isolated recovery, and product purity must be evaluated separately. By distinguishing PN fermentation, intracellular cofactor supply, PNP intermediates, reaction-broth PLP concentration, and isolated PLP yield, this review proposes a route-readiness map for PLP-producing cascades. The most promising next-generation systems will couple product-tolerant phosphorylation, oxidase performance, cofactor regeneration, and downstream stabilization in a single process-aware design. Full article

Background/Objectives: Aromatic L-amino acid decarboxylase deficiency (AADC-D; OMIM #608643) is a rare autosomal recessive neurometabolic disorder caused by pathogenic variants in the DDC gene, leading to impaired of monoamine neurotransmitter biosynthesis. AADC, a pyridoxal-5′-phosphate (PLP)-dependent enzyme, catalyzes the conversion of L-dopa and 5-hydroxytryptophan (5-HTP) to dopamine and serotonin, respectively. Early diagnosis remains challenging due to the limited specificity of current biochemical approaches. This study aimed to evaluate plasma AADC enzyme activity using these physiological substrates by High-Performance Liquid Chromatography (HPLC)-based method and assess its potential utility in the biochemical diagnosis of AADC deficiency. Methods: Plasma AADC activity was quantified using physiological substrates (L-dopa and 5-HTP) by HPLC with electrochemical and fluorescence detection. Sanger sequencing of the DDC gene was performed in two suspected patients to identify pathogenic variants. Results: Two genetically confirmed AADC-D patients demonstrated reduced enzyme activity. Using L-dopa as substrate, enzyme activity in patients was 12.4 and 26.1 pmol/min/mL, both below the published reference interval (36–129 pmol/min/mL). Using 5-HTP as substrate, enzyme activity was 1.5 and 5.1 pmol/min/mL; Patient 1 showed activity below the reference interval (2.0–7.1 pmol/min/mL), while Patient 2 demonstrated activity within the lower range of reported values. Reduced enzyme activity was consistent with the clinical features and molecular findings with identification of pathogenic variants in the DDC gene (c.175G>A and c.714+4A>T). Conclusions: Plasma AADC activity measurement demonstrates potential as a functional biochemical biomarker that augments molecular genetic testing in the biochemical evaluation of AADC deficiency. Further studies involving larger patient cohorts are required to further evaluate its diagnostic performance and broader clinical applicability. Full article

Threonine aldolases (TAs) are pyridoxal-5-phosphate (PLP)-dependent enzymes that catalyze the reversible aldol condensation between aldehydes and glycine, enabling the asymmetric synthesis of chiral β-hydroxy-α-amino acids. The discovery of new TAs has greatly enriched the enzyme family and spurred investigations into noncanonical reactions beyond conventional aldol condensation, establishing access to structurally diverse amino compounds. Protein engineering has significantly improved the catalytic performance of this enzyme class, particularly addressing the inherent limitation of low C_β-stereoselectivity. Furthermore, the integration of TAs into multi-enzyme cascade systems has broadened their synthetic applicability. This review systematically presents recent progress in biocatalysis with TAs, including the various reaction types, the engineering of TAs for improved catalytic performance, the application of the enzyme in cascade reactions, and future opportunities to broaden the utility of the enzyme class in amino compound synthesis. Full article

Accumulating evidence suggests that vitamin B6 (B6) deficiency among older adults is associated with sarcopenia, frailty, heart disease, and brain diseases. Oxidative stress and inflammation play key roles in cardiac and skeletal muscle and neuronal pathology. However, the detailed roles of B6 supplementation in oxidative stress and inflammation are not fully understood. Recent studies have shown that supplemental B6 upregulated the nuclear factor erythroid 2-like 2 (Nrf2) signaling pathway with the coordinated activation of antioxidant responses. Accumulating evidence suggests the potential of targeted Nrf2 signaling regulation in the treatment of aging-related musculoskeletal, heart, and brain diseases. Notably, dietary supplementation of B6 elevates the levels of several antioxidant metabolites, such as carnosine, anserine, taurine, hydrogen sulfide (H₂S), 5-methyltetrahydrofolate, kynurenic acid, 3-hydroxyanthranilic acid, and γ-aminobutyric acid (GABA) via the upregulation of pyridoxal 5′-phosphate (PLP)-dependent metabolic pathways, thereby linking to Nrf2 signaling activation. Furthermore, supplemental B6 stimulates glycogen breakdown through the PLP enzyme, glycogen phosphorylase, which in turn enhances the pentose phosphate pathway, thereby increasing nicotinamide adenine dinucleotide phosphate (NADPH) availability to regenerate glutathione (GSH). In this perspective article, we propose the potential role of B6 as an antioxidant mediated by the PLP-dependent multi-metabolic productions of antioxidant metabolites. Full article

Diagnosis and monitoring of type 2 diabetes mellitus (T2DM) typically rely on invasive blood-based biomarkers. To explore non-invasive alternatives, this study examined tongue coating metabolites to identify metabolic signatures linked to diabetes progression. A case-control observational study categorized participants into control, prediabetes, and diabetes groups. Tongue coating samples were analyzed using liquid chromatography-mass spectrometry (LC-MS). Differential metabolites were correlated with clinical parameters, including HbA1c, BMI, and eGFR. Distinct metabolic profiles emerged across groups, with significant differences in five endogenous metabolites (phenylpyruvic acid, propionylcarnitine, pyridoxal 5′-phosphate, phenethylamine, phenethylamine glucuronide) and four amino acids (isoleucine, lysine, phenylalanine, tyrosine). Diabetic subjects showed elevated phenylpyruvic acid and phenethylamine, while propionylcarnitine, pyridoxal 5′-phosphate, and phenethylamine glucuronide were reduced. Phenethylamine was positively correlated with HbA1c; propionylcarnitine and phenethylamine glucuronide showed negative correlations with HbA1c and BMI. Detected total amino acids were inversely correlated with eGFR. Additionally, a diabetes index derived from these metabolic features also holds potential for discriminating disease states. These findings underscore the potential of tongue coating metabolites as a relatively non-invasive approach for evaluating T2DM states. The observed metabolic alterations provide valuable insights into diabetes-associated dysregulation, including protein glycation, obesity-related metabolic shifts, and renal impairment. Full article

Pyridoxine-dependent epilepsy due to ALDH7A1 deficiency (PDE-ALDH7A1) is a rare but treatable epileptic encephalopathy caused by disruption of lysine catabolism and secondary depletion of pyridoxal-5′-phosphate (PLP). Although seizures are often controlled with pyridoxine supplementation, many patients continue to experience neurodevelopmental impairment, underscoring the importance of early diagnosis and improved therapeutic strategies. Central to both diagnosis and pathophysiology is the accumulation of lysine-derived metabolites, most notably α-aminoadipate semialdehyde (α-AASA), its cyclic Schiff base Δ¹-piperideine-6-carboxylate (P6C), and pipecolic acid. These metabolites have become the biochemical hallmarks of PDE-ALDH7A1, linking ALDH7A1 pathogenic variants to PLP inactivation and neuronal dysfunction. However, their chemical instability and analytical requirements pose challenges for universal diagnostics and newborn screening. This review summarizes current understanding of lysine catabolism in health and disease, critically evaluates the diagnostic utility and limitations of classical biomarkers, and discusses emerging insights into their pathophysiological roles. We further highlight recent discoveries of novel, chemically stable biomarkers, including 6-oxopiperidine-2-carboxylic acid (6-oxo-PIP), 2-oxopropylpiperidine-2-carboxylic acid (2-OPP), and 6-hydroxy-2-aminocaproic acid (HACA), identified through advanced metabolomics approaches. These metabolites show promise for newborn screening and provide new mechanistic links between metabolic stress, seizure susceptibility, and ongoing neurological morbidity despite pyridoxine treatment. Collectively, advances in biomarker discovery are reshaping diagnostic strategies for PDE-ALDH7A1 and offering new perspectives on disease mechanisms, paving the way for earlier detection and the development of more effective, mechanism-based therapies. Full article

Vitamin B6 is an essential micronutrient whose biologically active form, pyridoxal 5′-phosphate (PLP), acts as a cofactor in metabolic reactions linked to tumorigenesis and also functions as an antioxidant. Low plasma PLP levels are consistently associated with cancer, but studies on dietary intake have yielded conflicting results. Overall, evidence suggests that the effects of vitamin B6 deficiency on cancer are context-dependent, varying with cell type and tumor stage. Accordingly, high expression of PDXK and PNPO, two key genes involved in PLP biosynthesis, is associated with tumor progression in some malignancies, whereas it correlates with improved outcomes in others. This review explores Drosophila melanogaster as a useful model to investigate underlying mechanisms, bypassing the limitations of human studies. Research in Drosophila demonstrates that PLP deficiency promotes cancer by triggering genomic instability. Furthermore, a critical PLP-SHMT gene–nutrient interaction impacting oncogenesis has been established in flies, offering significant therapeutic implications. Finally, studies in Drosophila have shown that PLP deficiency can promote tumor development by also triggering the loss of heterozygosity (LOH). These findings highlight Drosophila as a powerful tool to elucidate the molecular pathways linking vitamin B6 deficiency to cancer. Full article

Background/Objectives: Gamma-aminobutyric acid (GABA) is a bioactive, non-proteinaceous amino acid with potential health benefits. Weissella cibaria UF-274 is an important lactic acid bacterium isolated from Balinese fermented sausage (urutan) with GABA-producing abilities. The aim of this study was to enhance GABA synthesis in skim milk as a basal substrate, as well as whole genome sequencing and analysis to evaluate the functionality and safety of the strain. Methods: A Box–Behnken response surface design was used to enhance GABA accumulation in skim milk. Results: The optimum conditions for GABA production were at concentrations of glucose of 23.91 g/L, monosodium glutamate concentrations of 2.32 g/L and pyridoxal-5′-phosphate at 46 μM. The genome assembly produced a high-quality draft with a 2.53 Mb circular chromosome and 2378 coding sequences. A whole genome analysis revealed that the strain possesses a glutamine amidotransferase (puuD-like) as an alternative route linked to the GABA pathway. AntiSMASH prediction results showed that the strain has two biosynthetic gene clusters including terpene and type III polyketide synthases. Several bioinformatic approaches predicted no antibiotic resistance genes, while van genes encoding vancomycin resistance were detected with low pathogen risk with one approach. Conclusions: Weissella cibaria UF-274 is a promising GABA producer with genomic evidence and a good candidate for functional food development. Full article

Pyridoxal 5′-phosphate (PLP), the active form of vitamin B6, is an essential cofactor, yet its industrial supply still relies largely on multi-step chemical synthesis. Here, using the industrial chassis Corynebacterium glutamicum ATCC 13032, we proposed and validated a strategy based on a minimal heterologous entry coupled to endogenous pathway continuation, resulting in a distinct PLP-producing route. Three engineered strains were constructed and compared: S1 expressing ecepd from Escherichia coli; S2 co-expressing ecepd plus ecpdxB from Escherichia coli (a minimal two-gene module); and S3 carrying an additional ecpdxA from Escherichia coli and smpdxJ from Sinorhizobium meliloti to form a four-gene module as a benchmark for heterologous reconstruction. The wild-type (WT) strain produced a basal PLP level of 10.6 mg/L. Overexpressing ecepd alone increased the titer to 40.4 mg/L (3.8-fold vs WT), whereas the minimal two-gene module in S2 yielded the highest PLP titer of 95.5 mg/L (9.0-fold vs WT; 136.0% higher than S1). Notably, the four-gene module (S3) reached 70.0 mg/L, which was 36.3% lower than S2 under matched conditions. These results indicated that introducing only a minimal two-gene entry could cooperate with the endogenous metabolic network of Corynebacterium glutamicum to establish a new and highly effective PLP biosynthetic route, with production performance exceeding that of a multi-gene heterologous reconstruction in the tested window. This work provides a low-burden and scalable framework for sustainable PLP biomanufacturing and motivates further optimization targeting the endogenous continuation steps and regulatory constraints. Full article

Background: Lyases are used in a wide scope of applications, making them invaluable tools in both industrial biotechnology and molecular biology. Many examples of lyases belong to the extensive family of pyridoxal 5′-phosphate (PLP)-dependent enzymes, which catalyze numerous reactions involved in amino acid metabolism, like tryptophan indole-lyase (Trpase or Tnase), tyrosine phenol-lyase (TPL), and methionine γ-lyase (MGL). Beyond their role in physiological processes, these lyases can also facilitate the synthesis of other biologically active products from non-canonical substrates. Objectives: Up till now there were only two C–S lyases known for the thiosulfinates’ biosynthesis from S-substituted L-cysteine sulfoxides—alliinase and MGL. Our study reveals for the first time that C–C lyases are capable of C–S lyase activity in reactions with S-alkyl, S-allyl and S-benzyl cysteine sulfoxides. Methods: We have compared the kinetic profiles of S-substituted L-cysteine sulfoxide degradation mediated by carbon–sulfur lyase MGL versus carbon–carbon lyases TPL and Trpase. Results: Among other S-alkyl-L-cysteine sulfoxides, petiveriin (S-benzyl-L-cysteine sulfoxide) was proven to be a substrate for all three enzymes. The potential utility of these enzymes in thiosulfinate production was supported by in vitro testing of enzyme-generated thiosulfinates against clinically relevant pathogens such as Candida albicans, Pseudomonas aeruginosa, and Staphylococcus aureus. Conclusions: Both C–S and C–C lyases—MGL, TPL, and Trpase—can be implemented for practical application in thiosulfinate synthesis. Full article

Background: Exclusive breastfeeding for the first 6 months of corrected age (CA) is recommended for premature infants with very low birth weight (<1500 g) (VLBW). B vitamins are essential for normal development and growth, including DNA methylation, and we investigated whether exclusive breastfeeding for the first 6 months provides an adequate cobalamin, folate, vitamin B6, and vitamin B2 status compared to additional or exclusive formula feeding from term to 12 months CA. Methods: In recruited infants born prematurely with VLBW, levels of vitamin B12 (cobalamin), folate, vitamin B6 (pyridoxal 5′-phosphate, PLP), vitamin B2 (riboflavin), and the metabolic markers, total homocysteine (tHcy) and methylmalonic acid (MMA), were determined at term (n = 35) and at 2 (n = 47), 6 (n = 48), and 12 months (n = 58) CA. Results: Only a minority of the infants were given multivitamin supplementation, and this was associated with higher PLP and riboflavin levels. One-third of the infants were exclusively breastfed to 6 months CA, and these had lower cobalamin, PLP, and riboflavin concentrations compared to formula-fed infants; additionally, 80% had plasma tHcy concentrations ≥ 6.5 µmol/L, indicative of cobalamin deficiency during the first 6 months CA. Serious deficiency of one or more B vitamins was evident in 3–9% of the infants at each time point during the first 12 months CA, more often in breastfed infants, but not exclusively. Conclusions: Exclusive breastfeeding with inadequate multivitamin supplementation and no specific recommendation for introduction of solid food does not provide an adequate B vitamin status in infants born prematurely with VLBW. Nutritional micronutrient recommendations must be improved, and regular evaluation of vitamin status should be implemented in the follow-up for premature VLBW infants. Full article

Pathological reduction in enzymatic activity of the tissue-non-specific alkaline phosphatase (TNSALP) is the molecular hallmark of hypophosphatasia (HPP), a group of rare inborn systemic diseases, mainly characterized by pathological affections of calcified tissue mineralization and the musculoskeletal system. The disease, in all clinical forms, is biochemically characterized by variable degrees of chronically reduced activity of circulating total alkaline phosphatase (ALP). Repeated detection of low values of ALP activity is mandatory to diagnose the presence of HPP, but, alone, it is not sufficient for the diagnosis of the disease. Detection of increased circulating levels of one of the main natural substrates of TNSALP, the pyridoxal 5′-phosphate (PLP), is needed to biochemically confirm the diagnosis of HPP. Urinary and/or blood levels of phosphoethanolamine (PEA) and inorganic pyrophosphate (PPi), two other natural substrates of TNSALP, can be elevated in a percentage of HPP patients. The contemporary biochemical evaluation of ALP activity and its target substrates is of great help in the diagnosis of HPP, and also for the monitoring of a patient’s response to enzymatic replacement therapy or other pharmacological treatments. Here, we describe and discuss possibilities and challenges of biochemical screenings for HPP, based also on the experience gained in our analysis laboratory. Full article

This study presents a comprehensive analysis of cysteine synthase A (CysK) from Limosilactobacillus reuteri LR1 (LreCysK), an enzyme involved in the biosynthesis of L-cysteine. This protein supports crucial cellular functions such as sulfur metabolism, antioxidant defense, detoxification, and protein synthesis. Previously, the gene encoding LreCysK was cloned, and the enzyme with His-tag on the N-terminus was obtained in active and soluble form. Here, kinetic parameters of the enzyme were determined by the previously developed high-pressure liquid chromatography (HPLC) and ninhydrin methods. It was found that LreCysK has similar K_M^OAS and k_cat as CysKs from Escherichia coli and from the model plant Arabidopsis thaliana. The thermal stability of LreCysK was studied using differential scanning calorimetry. It was revealed that the melting point of the enzyme increases to almost 90°C when Pyridoxal-5 phosphate (PLP) is added, indicating that the stability of the enzyme complex with PLP is relatively high. Structural studies revealed that LreCysK is a dimer, and its active site is similar to those of other enzymes, but exhibits some features characteristic of lactobacilli CysKs (GISA), as well as unique residues, such as Ile50. Also, the potential biotechnological applications of LreCysK are discussed. These findings enhance our understanding of LreCysK’s biochemical versatility and its potential applications in biotechnology and medicine. Full article