This chapter presents a method for the heterologous expression and purification of human ALA synthase from Escherichia coli. Mature ALAS is produced with an N-terminal hexahistidine affinity tag followed by a SUMO fusion tag for solubility and ease of purification. The plasmid is introduced into competent E. coli cells, and robust protein expression is induced with IPTG. The ALAS cofactor, pyridoxal 5\'-phosphate, is inserted during protein production to yield an active enzyme upon purification. After cell lysis, the tagged ALAS protein is isolated via a multistep purification that involves an initial nickel-affinity step, affinity tag cleavage and removal, and a final size exclusion chromatography polishing step. Importantly, this protocol is amenable to various ALAS truncations and mutations, opening the door to understanding ALAS biology and its intersections with iron utilization across several organisms.

Naturally occurring forms of vitamin B6 include six interconvertible water-soluble compounds: pyridoxine (PN), pyridoxal (PL), pyridoxamine (PM), and their respective monophosphorylated derivatives (PNP, PLP, and PMP). PLP is the catalytically active form which works as a cofactor in approximately 200 reactions that regulate the metabolism of glucose, lipids, amino acids, DNA, and neurotransmitters. Most of vitamers can counteract the formation of reactive oxygen species and the advanced glycation end-products (AGEs) which are toxic compounds that accumulate in diabetic patients due to prolonged hyperglycemia. Vitamin B6 levels have been inversely associate with diabetes, while vitamin B6 supplementation reduces diabetes onset and its vascular complications. The mechanisms at the basis of the relation between vitamin B6 and diabetes onset are still not completely clarified. In contrast more evidence indicates that vitamin B6 can protect from diabetes complications through its role as scavenger of AGEs. It has been demonstrated that in diabetes AGEs can destroy the functionality of macromolecules such as protein, lipids, and DNA, thus producing tissue damage that result in vascular diseases. AGEs can be in part also responsible for the increased cancer risk associated with diabetes. In this chapter the relationship between vitamin B6, diabetes and AGEs will be discussed by showing the acquired knowledge and questions that are still open.

Hypophosphatasia (HPP) is the dento-osseous disorder caused by deactivating mutation(s) of ALPL, the gene that encodes the \"tissue-nonspecific\" isoenzyme of alkaline phosphatase (TNSALP). In HPP, 3 natural substrates of cell-surface TNSALP accumulate extracellularly; phosphoethanolamine (PEA), inorganic pyrophosphate (PPi), and pyridoxal 5\'-phosphate (PLP). Hypophosphatasemia together with elevated plasma levels of PEA, PPi, and PLP comprise its biochemical signature. PPi can inhibit mineralization and in extracellular excess can impair bone and tooth hardening and perhaps explain weak muscle. Autosomal dominant or autosomal recessive inheritance from among more than 400 mutations of ALPL largely accounts for HPP\'s broad-ranging severity, greatest among all skeletal diseases. Pediatric HPP spans life-threatening perinatal and infantile forms, childhood forms, and odonto-HPP selectively featuring premature loss of deciduous teeth. ALPL gene testing and TNSALP supplementation therapy have bolstered familiarity with HPP, but there are new considerations for diagnosis. Herein, diagnosis of a boy\'s mild childhood HPP was delayed by missteps involving his medical and dental history, physical examination, radiographic findings, and clinical laboratory studies. We review how pediatric HPP is now identified. Prompt diagnosis while appreciating the broad-ranging severity of HPP underlies the safe and effective management of this inborn-error-of-metabolism.

A case of an adult with borderline AADC deficiency symptoms is presented here. Genetic analysis revealed that the patient carries two AADC variants (NM_000790.3: c.1040G > A and c.679G > C) in compound heterozygosis, resulting in p.Arg347Gln and p.Glu227Gln amino acid alterations. While p.Arg347Gln is a known pathogenic variant, p.Glu227Gln is unknown. Combining clinical features to bioinformatic and molecular characterization of the AADC protein population of the patient (p.Arg347Gln/p.Arg347Gln homodimer, p.Glu227Gln/p.Glu227Gln homodimer, and p.Glu227Gln/p.Arg347Gln heterodimer), we determined that: i) the p.Arg347Gln/p.Arg347Gln homodimer is inactive since the alteration affects a catalytically essential structural element at the active site, ii) the p.Glu227Gln/p.Glu227Gln homodimer is as active as the wild-type AADC since the alteration occurs at the surface and does not change the chemical nature of the amino acid, and iii) the p.Glu227Gln/p.Arg347Gln heterodimer has a catalytic efficiency 75% that of the wild-type since only one of the two active sites is compromised, thus demonstrating a positive complementation. By this approach, the molecular basis for the mild presentation of the disease is provided, and the experience made can also be useful for personalized therapeutic decisions in other mild AADC deficiency patients. Interestingly, in the last few years, many previously undiagnosed or misdiagnosed patients have been identified as mild cases of AADC deficiency, expanding the phenotype of this neurotransmitter disease.

Aromatic amino acid decarboxylase is a pyridoxal 5\'-phosphate-dependent enzyme responsible for the synthesis of the neurotransmitters, dopamine and serotonin. Here, by a combination of bioinformatic predictions and analyses, phosphorylation assays, spectroscopic investigations and activity measurements, we determined that Ser-193, a conserved residue located at the active site, can be phosphorylated, increasing catalytic efficiency. In order to determine the molecular basis for this functional improvement, we determined the structural and kinetic properties of the site-directed variants S193A, S193D and S193E. While S193A retains 27% of the catalytic efficiency of wild-type, the two acidic side chain variants are impaired in catalysis with efficiencies of about 0.15% with respect to the wild-type. Thus, even if located at the active site, Ser-193 is not essential for enzyme activity. We advance the idea that this residue is fundamental for the correct architecture of the active site in terms of network of interactions triggering catalysis. This role has been compared with the properties of the Ser-194 of the highly homologous enzyme histidine decarboxylase whose catalytic loop is visible in the spatial structure, allowing us to propose the validation for the effect of the phosphorylation. The effect could be interesting for AADC deficiency, a rare monogenic disease, whose broad clinical phenotype could be also related to post translational AADC modifications.

The Gram-negative bacterium, Fusobacterium nucleatum, possesses a fold II type pyridoxal 5\'-phosphate-dependent enzyme that catalyzes the reversible β-replacement of l-cysteine and l-serine, generating H2S and H2O, respectively. This enzyme, termed serine synthase (FN1055), contains an active site Asp232 that serves as a general base in the activation of a water molecule for nucleophilic attack of the ⍺-aminoacrylate intermediate. A network of hydrophobic residues surrounding Asp232 are key to catalysis as they increase the basicity of the side chain. However, these residues severely restrict the range of nucleophilic substrates that can react with the ⍺-aminoacrylate, making the enzyme an ineffective biocatalyst for noncanonical amino acid biosynthesis. Herein, we systematically substituted four aromatic active residues (Trp99, Phe125, Phe148 and Phe234) to an alanine to determine their catalytic importance in serine/cysteine synthase reactions and if their substitution could broaden the scope of nucleophiles that could react with the ⍺-aminoacrylate intermediate. All four single site mutants W99A, F125A, F148A, and F234A could form the ⍺-aminoacrylate intermediate upon reaction with either l-cysteine or l-serine; however, the rate constant associated with the elimination of the β-hydroxyl group from l-serine was 150 to 200-fold lower in the F125A and F148A variants. Substitution of Phe125 and Phe148, situated ∼3-4 Å from the general base, also abolished the serine synthase reaction due to their inability to activate a water molecule for nucleophilic attack of the ⍺-aminoacrylate. Overall, the mutational studies indicate that the clustering of aromatic residues disproportionately benefits the serine synthase reaction as they increase the binding affinity for l-cysteine, decrease the binding of the product, l-serine, and promote the activation of a water molecule. Notably, the aminoacrylate species present in F125A and F148A was able to react with thiophenol, signifying that serine synthase has biocatalytic potential in the synthesis of noncanonical amino acids.

Over the past two decades, the field of vitamin B6 -dependent epilepsies has evolved by the recognition of a growing number of gene defects (ALDH7A1, PNPO, ALPL, ALDH4A1, PLPBP as well as defects of the glycosylphosphatidylinositol anchor proteins) that all lead to reduced availability of pyridoxal 5\'-phosphate, an important cofactor in neurotransmitter and amino acid metabolism. In addition, positive pyridoxine response has been observed in other monogenic defects such as MOCS2 deficiency or KCNQ2 and there may be more defects to be discovered. Most entities lead to neonatal onset pharmaco-resistant myoclonic seizures or even status epilepticus and pose an emergency to the treating physician. Research has unraveled specific biomarkers for several of these entities (PNPO deficiency, ALDH7A1 deficiency, ALDH4A1 deficiency, ALPL deficiency causing congenital hypophosphatasia and glycosylphosphatidylinositol anchoring defects with hyperphosphatasia), that can be detected in plasma or urine, while there is no biomarker to test for PLPHP deficiency. Secondary elevation of glycine or lactate was recognized as diagnostic pitfall. An algorithm for a standardized trial with vitamin B6 should be in place in every newborn unit in order not to miss these well-treatable inborn errors of metabolism. The Komrower lecture of 2022 provided me with the opportunity to tell the story about the conundrums of research into vitamin B6 -dependent epilepsies that kept some surprises and many novel insights into pathomechanisms of vitamin metabolism. Every single step had benefits for the patients and families that we care for and advocates for a close collaboration of clinician scientists with basic research.

We describe the photoinduced reductive debromination of phenacyl bromides using pyridoxal 5\'-phosphate (PLP). The reaction requires irradiation with cyan or blue light in an anaerobic atmosphere. Mechanistic analysis reveals the formation of the phenacyl radical as an intermediate in the reaction, implying a single electron transfer to phenacyl bromides from a PLP-derived species resulting from excitation by illumination.

Gold(III) complexes with different ligands can provide researchers with a measure against pathogenic microorganisms with antibiotic resistance. We reported in our previous paper that the UV-Vis spectra of different protonated species of complexes formed by gold(III) and five hydrazones derived from pyridoxal 5\'-phosphate are similar to each other and to the spectra of free protonated hydrazones. The present paper focuses on the reasons of the noted similarity in electron absorption spectra. The geometry of different protonated species of complexes of gold(III) and hydrazones (15 structures in total) was optimized using the density functional theory (DFT). The coordination polyhedron of gold(III) bond critical points were further studied to identify the symmetry of the gold coordination sphere and the type of interactions that hold the complex together. The UV-Vis spectra were calculated using TD DFT methods. The molecular orbitals were analyzed to interpret the calculated spectra.

Vitamin B6 is an indispensable micronutrient in organisms and is widely distributed in blood, tissues, and organs. Changes in the content and ratio of vitamin B6 can affect the entire physiological condition of the body, so it becomes particularly important to reveal the relationship between changes in its content and disease by monitoring vitamin B6 levels in the organism. In this study, a two-dimensional liquid chromatography-UV detector (2D-LC-UV) was used to establish a method for the simultaneous detection of PLP, PA, and PL for the first time. First, PLP, PA, and PL were extracted with plasma: 0.6 M TCA: ultrapure water = 1:2:3 (v/v/v) and then derivatized. Enrichment and preliminary separation were performed on a one-dimensional column and automatically entered into a two-dimensional column for further separation. This method exhibited good selectivity, and the correlation coefficients for the analyte calibration curves were >0.99. The detection limits for PLP, PA, and PL were 0.1, 0.2, and 4 nmol/L, respectively. The results showed that the system has high loading capacity, excellent resolution, and a good peak shape. This method is expected to provide applicability for the determination of PLP, PA, and PL in pharmacological, pharmaceutical, and clinical research.