Enzymes that use the cofactor pyridoxal phosphate (PLP) constitute a ubiquitous class of biocatalysts. Here, we analyse their variety and genomic distribution as an example of the current opportunities and challenges for the study of protein families. In many free-living prokaryotes, almost 1.5% of all genes code for PLP-dependent enzymes, but in higher eukaryotes the percentage is substantially lower, consistent with these catalysts being involved mainly in basic metabolism. Assigning the function of PLP-dependent enzymes simply on the basis of sequence criteria is not straightforward because, as a consequence of their common mechanistic features, these enzymes have intricate evolutionary relationships. Thus, many genes for PLP-dependent enzymes remain functionally unclassified, and several of them might encode undescribed catalytic activities. In addition, PLP-dependent enzymes often show catalytic promiscuity (that is, a single enzyme catalyses different reactions), implying that an organism can have more PLP-dependent activities than it has genes for PLP-dependent enzymes. This observation presumably applies to many other classes of protein-encoding genes.

Pyridoxal-5'-phosphate (PLP) is a versatile cofactor that assists in different types of enzymatic reactions. PLP has also been reported to react with substrates and catalyze some of these reactions independent of enzymes. One such catalytic reaction is the breakdown of cysteine to produce hydrogen sulfide (H2S) in the presence of multivalent metal ions. However, the enzyme-independent catalytic activity of PLP in catabolizing cysteine in the absence of multivalent ions is unknown. In this study, we show that PLP reacts with cysteine to form a thiazolidine product, which is supported by quantum chemical calculations of the absorption spectrum. The reaction of PLP with cysteine is dependent on ionic strength and pH. The thiazolidine product slowly decomposes to produce H2S and the PLP regenerates to its active form with longer reaction times (> 24 h), suggesting that PLP can act as a catalyst. We propose an enzyme-independent plausible reaction mechanism for PLP catalyzed cysteine breakdown to produce H2S, which proceeds through the formation of thiazolidine ring intermediates that later hydrolyzes slowly to regenerate the PLP. This work demonstrates that PLP catalyzes cysteine breakdown in the absence of enzymes, base, and multivalent metal ions to produce H2S.

To identify disease-causing variants in autosomal recessive axonal polyneuropathy with optic atrophy and provide targeted replacement therapy.

Several nucleoside antibiotics are structurally characterized by a 5″-amino-5″-deoxyribose (ADR) appended via a glycosidic bond to a high-carbon sugar nucleoside (5'S,6'S)-5'-C-glycyluridine (GlyU). GlyU is further modified with an N-alkylamine linker, the biosynthetic origin of which has yet to be established. By using a combination of feeding experiments with isotopically labeled precursors and characterization of recombinant proteins from multiple pathways, the biosynthetic mechanism for N-alkylamine installation for ADR-GlyU-containing nucleoside antibiotics has been uncovered. The data reveal S-adenosyl-L-methionine (AdoMet) as the direct precursor of the N-alkylamine, but, unlike conventional AdoMet- or decarboxylated AdoMet-dependent alkyltransferases, the reaction is catalyzed by a pyridoxal-5'-phosphate-dependent aminobutyryltransferase (ABTase) using a stepwise γ-replacement mechanism that couples γ-elimination of AdoMet with aza-γ-addition onto the disaccharide alkyl acceptor. In addition to using a conceptually different strategy for AdoMet-dependent alkylation, the newly discovered ABTases require a phosphorylated disaccharide alkyl acceptor, revealing a cryptic intermediate in the biosynthetic pathway.

The anticancer actions of the biguanide metformin involve the functioning of the serine/glycine one-carbon metabolic network. We report that metformin directly and specifically targets the enzymatic activity of mitochondrial serine hydroxymethyltransferase (SHMT2). In vitro competitive binding assays with human recombinant SHMT1 and SHMT2 isoforms revealed that metformin preferentially inhibits SHMT2 activity by a non-catalytic mechanism. Computational docking coupled with molecular dynamics simulation predicted that metformin could occupy the cofactor pyridoxal-5'-phosphate (PLP) cavity and destabilize the formation of catalytically active SHMT2 oligomers. Differential scanning fluorimetry-based biophysical screening confirmed that metformin diminishes the capacity of PLP to promote the conversion of SHMT2 from an inactive, open state to a highly ordered, catalytically competent closed state. CRISPR/Cas9-based disruption of SHMT2, but not of SHMT1, prevented metformin from inhibiting total SHMT activity in cancer cell lines. Isotope tracing studies in SHMT1 knock-out cells confirmed that metformin decreased the SHMT2-channeled serine-to-formate flux and restricted the formate utilization in thymidylate synthesis upon overexpression of the metformin-unresponsive yeast equivalent of mitochondrial complex I (mCI). While maintaining its capacity to inhibit mitochondrial oxidative phosphorylation, metformin lost its cytotoxic and antiproliferative activity in SHMT2-null cancer cells unable to produce energy-rich NADH or FADH2 molecules from tricarboxylic acid cycle (TCA) metabolites. As currently available SHMT2 inhibitors have not yet reached the clinic, our current data establishing the structural and mechanistic bases of metformin as a small-molecule, PLP-competitive inhibitor of the SHMT2 activating oligomerization should benefit future discovery of biguanide skeleton-based novel SHMT2 inhibitors in cancer prevention and treatment.

Pyridoxal 5'-phosphate (PLP) is the most important cofactor of vitamin B₆-dependent enzymes, which catalyses a wide range of essential body functions (e.g., metabolism) that could be exploited to specifically target highly metabolic cells, such as tumour metastatic cells. However, the use of PLP as a simultaneous coating and targeting molecule, which at once provides colloidal stability and specific biological effects has not been exploited so far. Therefore, in this work iron oxide nanoparticles (IONPs) were coated by PLP at two different pH values to tune PLP bonding (e.g., orientation) at the IONP surface. The surface study, as well as calculations, confirmed different PLP bonding to the IONP surface at these two pH values. Moreover, the obtained PLP-IONPs showed different zeta potential, hydrodynamic radius and agglomeration state, and consequently different uptake by two metastatic-prostate-cancer cell lines (LnCaP and PC3). In LnCaP cells, PLP modified the morphology of IONP-containing intracellular vesicles, while in PC3 cells PLP impacted the amount of IONPs taken up by cells. Moreover, PLP-IONPs displayed high magnetic resonance imaging (MRI) r₂ relaxivity and were not toxic for the two studied cell lines, rendering PLP promising for biomedical applications. We here report the use of PLP simultaneously as a coating and targeting molecule, directly bound to the IONP surface, with the additional high potential for MRI detection.

A set of phosphonic acid derivatives (1-4) of pyridoxal 5'-phosphate (PLP) was synthesized and characterized biochemically using purified murine pyridoxal phosphatase (PDXP), also known as chronophin. The most promising compound 1 displayed primarily competitive PDXP inhibitory activity with an IC50 value of 79μM, which was in the range of the Km of the physiological substrate PLP. We also report the X-ray crystal structure of PDXP bound to compound 3, which we solved to 2.75Å resolution (PDB code 5AES). The co-crystal structure proves that compound 3 binds in the same orientation as PLP, and confirms the mode of inhibition to be competitive. Thus, we identify compound 1 as a PDXP phosphatase inhibitor. Our results suggest a strategy to design new, potent and selective PDXP inhibitors, which may be useful to increase the sensitivity of tumor cells to treatment with cytotoxic agents.

The concentrations of pyridoxal 5'-phosphate in the brains of DBA/2J mice during the withdrawal period following chronic ethanol ingestion were measured fluorimetrically. The brain of the ethanol-withdrawn mouse contains 22.4 +/- 3.1% more pyridoxal 5'-phosphate than the brain of a control mouse. The enhanced susceptibility to epileptic seizures during the withdrawal phase may in part be due to the increased cerebral content of this coenzyme.

The MocR-subfamily transcription factors (MocR-TFs) characterized by the GntR-family DNA-binding domain and aminotransferase-like sensory domain are broadly distributed among certain lineages of Bacteria. Characterized MocR-TFs bind pyridoxal 5'-phosphate (PLP) and control transcription of genes involved in PLP, gamma aminobutyric acid (GABA) and taurine metabolism via binding specific DNA operator sites. To identify putative target genes and DNA binding motifs of MocR-TFs, we performed comparative genomics analysis of over 250 bacterial genomes. The reconstructed regulons for 825 MocR-TFs comprise structural genes from over 200 protein families involved in diverse biological processes. Using the genome context and metabolic subsystem analysis we tentatively assigned functional roles for 38 out of 86 orthologous groups of studied regulators. Most of these MocR-TF regulons are involved in PLP metabolism, as well as utilization of GABA, taurine and ectoine. The remaining studied MocR-TF regulators presumably control genes encoding enzymes involved in reduction/oxidation processes, various transporters and PLP-dependent enzymes, for example aminotransferases. Predicted DNA binding motifs of MocR-TFs are generally similar in each orthologous group and are characterized by two to four repeated sequences. Identified motifs were classified according to their structures. Motifs with direct and/or inverted repeat symmetry constitute the majority of inferred DNA motifs, suggesting preferable TF dimerization in head-to-tail or head-to-head configuration. The obtained genomic collection of in silico reconstructed MocR-TF motifs and regulons in Bacteria provides a basis for future experimental characterization of molecular mechanisms for various regulators in this family.

Pyridoxal 5'-phosphate (PLP), the catalytically active form of vitamin B6, plays a pivotal role in metabolism as an enzyme cofactor. PLP is a very reactive molecule and can be very toxic unless its intracellular concentration is finely regulated. In Escherichia coli, PLP formation is catalyzed by pyridoxine 5'-phosphate oxidase (PNPO), a homodimeric FMN-dependent enzyme that is responsible for the last step of PLP biosynthesis and is also involved in the PLP salvage pathway. We have recently observed that E. coli PNPO undergoes an allosteric feedback inhibition by PLP, caused by a strong allosteric coupling between PLP binding at the allosteric site and substrate binding at the active site. Here we report the crystallographic identification of the PLP allosteric site, located at the interface between the enzyme subunits and mainly circumscribed by three arginine residues (Arg23, Arg24, and Arg215) that form an "arginine cage" and efficiently trap PLP. The crystal structure of the PNPO-PLP complex, characterized by a marked structural asymmetry, presents only one PLP molecule bound at the allosteric site of one monomer and sheds light on the allosteric inhibition mechanism that makes the enzyme-substrate-PLP ternary complex catalytically incompetent. Site-directed mutagenesis studies focused on the arginine cage validate the identity of the allosteric site and provide an effective means to modulate the allosteric properties of the enzyme, from the loosening of the allosteric coupling (in the R23L/R24L and R23L/R215L variants) to the complete loss of allosteric properties (in the R23L/R24L/R21L variant).

The pyridoxal 5'-phosphate-binding protein (PLPBP) is an evolutionarily conserved protein linked to pyridoxal 5'-phosphate-binding. Although mutations in PLPBP were shown to cause vitamin B6-dependent epilepsy, its cellular role and function remain elusive. We here report a detailed biochemical investigation of human PLPBP and its epilepsy-causing mutants by evaluating stability, cofactor binding, and oligomerization. In this context, chemical cross-linking combined with mass spectrometry unraveled an unexpected dimeric assembly of PLPBP. Furthermore, the interaction network of PLPBP was elucidated by chemical cross-linking paired with co-immunoprecipitation. A mass spectrometric analysis in a PLPBP knockout cell line resulted in distinct proteomic changes compared to wild type cells, including upregulation of several cytoskeleton- and cell division-associated proteins. Finally, transfection experiments with vitamin B6-dependent epilepsy-causing PLPBP variants indicate a potential role of PLPBP in cell division as well as proper muscle function. Taken together, our studies on the structure and cellular role of human PLPBP enable a better understanding of the physiological and pathological mechanism of this important protein.

Enzymes dependent on pyridoxal 5'-phosphate (PLP, the active form of vitamin B6) perform a myriad of diverse chemical transformations. They promote various reactions by modulating the electronic states of PLP through weak interactions in the active site. Neutron crystallography has the unique ability of visualizing the nuclear positions of hydrogen atoms in macromolecules. Here we present a room-temperature neutron structure of a homodimeric PLP-dependent enzyme, aspartate aminotransferase, which was reacted in situ with α-methylaspartate. In one monomer, the PLP remained as an internal aldimine with a deprotonated Schiff base. In the second monomer, the external aldimine formed with the substrate analog. We observe a deuterium equidistant between the Schiff base and the C-terminal carboxylate of the substrate, a position indicative of a low-barrier hydrogen bond. Quantum chemical calculations and a low-pH room-temperature X-ray structure provide insight into the physical phenomena that control the electronic modulation in aspartate aminotransferase.Pyridoxal 5'-phosphate (PLP) is a ubiquitous co factor for diverse enzymes, among them aspartate aminotransferase. Here the authors use neutron crystallography, which allows the visualization of the positions of hydrogen atoms, and computation to characterize the catalytic mechanism of the enzyme.

Pyridoxal phosphate (PLP) is an enzyme cofactor required for the chemical transformation of biological amines in many central cellular processes. PLP-dependent enzymes (PLP-DEs) are ubiquitous and evolutionarily diverse, making their classification based on sequence homology challenging. Here we present a chemical proteomic method for reporting on PLP-DEs using functionalized cofactor probes. We synthesized pyridoxal analogues modified at the 2'-position, which are taken up by cells and metabolized in situ. These pyridoxal analogues are phosphorylated to functional cofactor surrogates by cellular pyridoxal kinases and bind to PLP-DEs via an aldimine bond which can be rendered irreversible by NaBH4 reduction. Conjugation to a reporter tag enables the subsequent identification of PLP-DEs using quantitative, label-free mass spectrometry. Using these probes we accessed a significant portion of the Staphylococcus aureus PLP-DE proteome (73%) and annotate uncharacterized proteins as novel PLP-DEs. We also show that this approach can be used to study structural tolerance within PLP-DE active sites and to screen for off-targets of the PLP-DE inhibitor D-cycloserine.

Pyridoxal 5'-phosphate (PLP) is a versatile cofactor that catalyzes a plethora of chemical transformations within a cell. Although many human PLP-dependent enzymes (PLP-DEs) with crucial physiological and pathological roles are known, a global method enabling their cellular profiling is lacking. Here, we demonstrate the utility of a cofactor probe for the identification of human PLP-binding proteins in living cells. Striking selectivity of human pyridoxal kinase led to a customized labeling strategy covering a large fraction of known PLP-binding proteins across various cancer-derived cell lines. Labeling intensities of some PLP-DEs varied depending on the cell type while the overall protein expression levels of these proteins remained constant. In addition, we applied the methodology for in situ screening of PLP-antagonists and unraveled known binders as well as unknown off-targets. Taken together, our proteome-wide method to study PLP-DEs in human cancer-derived cells enables global understanding of the interactome of this important cofactor.

To investigate the utility of serum pyridoxal 5'-phosphate (PLP), pyridoxal (PL), and 4-pyridoxic acid (PA) as a diagnostic marker of hypophosphatasia (HPP) and an indicator of the effect of, and patient compliance with, enzyme replacement therapy (ERT), we measured PLP, PL, and PA concentrations in serum samples from HPP patients with and without ERT.

Pyridaben (PY) is a widely used organochlorine acaricide, which can be detected in the peripheral blood of pregnant women. Available evidence suggests that PY has reproductive toxicity. However, it remains uncertain whether prenatal PY exposure impacts neurobehavioral development in offspring. Here, we administered PY to pregnant mice at a dose of 0.5 and 5 mg kg-1 day-1 via gavage and observed anxiety-like behaviors in PY offspring aged five weeks. We then integrated the metabolome and transcriptome of the offspring's brain to explore the underlying mechanism. Metabolome data indicated that the vitamin B6 metabolism pathway was significantly affected, and the pyridoxal 5'-phosphate (PLP) concentration and the active form of vitamin B6 was significantly reduced. Moreover, the transcriptome data showed that both PLP generation-related Pdxk and anxiety-related Gad1 were significantly down-regulated. Meanwhile, there was a decreasing trend in the concentration of GABA in the hippocampal DG region. Next, we supplemented PLP at a dose of 20 mg kg-1 day-1 to the PY offspring via intraperitoneal injection at three weeks. We found up-regulated expression of Pdxk and Gad1 and restored anxiety-like behaviors. This study suggests that prenatal exposure to PY can disrupt vitamin B6 metabolism, reduce the concentration of PLP, down-regulate the expression levels of Pdxk and Gad1, inhibit the production of GABA, and ultimately lead to anxiety-like behaviors in offspring.

Cobalamin-dependent enzymes enhance the rate of C-Co bond cleavage by up to ∼10(12)-fold to generate cob(II)alamin and a transient adenosyl radical. In the case of the pyridoxal 5'-phosphate (PLP) and cobalamin-dependent enzymes lysine 5,6-aminomutase and ornithine 4,5 aminomutase (OAM), it has been proposed that a large scale domain reorientation of the cobalamin-binding domain is linked to radical catalysis. Here, OAM variants were designed to perturb the interface between the cobalamin-binding domain and the PLP-binding TIM barrel domain. Steady-state and single turnover kinetic studies of these variants, combined with pulsed electron-electron double resonance measurements of spin-labeled OAM were used to provide direct evidence for a dynamic interface between the cobalamin and PLP-binding domains. Our data suggest that following ligand binding-induced cleavage of the Lys(629)-PLP covalent bond, dynamic motion of the cobalamin-binding domain leads to conformational sampling of the available space. This supports radical catalysis through transient formation of a catalytically competent active state. Crucially, it appears that the formation of the state containing both a substrate/product radical and Co(II) does not restrict cobalamin domain motion. A similar conformational sampling mechanism has been proposed to support rapid electron transfer in a number of dynamic redox systems.

Pyridoxal 5'-phosphate (PLP), the active form of vitamin B6, functions as a cofactor in humans for more than 140 enzymes, many of which are involved in neurotransmitter synthesis and degradation. A deficiency of PLP can present, therefore, as seizures and other symptoms that are treatable with PLP and/or pyridoxine. Deficiency of PLP in the brain can be caused by inborn errors affecting B6 vitamer metabolism or by inactivation of PLP, which can occur when compounds accumulate as a result of inborn errors of other pathways or when small molecules are ingested. Whole-exome sequencing of two children from a consanguineous family with pyridoxine-dependent epilepsy revealed a homozygous nonsense mutation in proline synthetase co-transcribed homolog (bacterial), PROSC, which encodes a PLP-binding protein of hitherto unknown function. Subsequent sequencing of 29 unrelated indivduals with pyridoxine-responsive epilepsy identified four additional children with biallelic PROSC mutations. Pre-treatment cerebrospinal fluid samples showed low PLP concentrations and evidence of reduced activity of PLP-dependent enzymes. However, cultured fibroblasts showed excessive PLP accumulation. An E.coli mutant lacking the PROSC homolog (ΔYggS) is pyridoxine sensitive; complementation with human PROSC restored growth whereas hPROSC encoding p.Leu175Pro, p.Arg241Gln, and p.Ser78Ter did not. PLP, a highly reactive aldehyde, poses a problem for cells, which is how to supply enough PLP for apoenzymes while maintaining free PLP concentrations low enough to avoid unwanted reactions with other important cellular nucleophiles. Although the mechanism involved is not fully understood, our studies suggest that PROSC is involved in intracellular homeostatic regulation of PLP, supplying this cofactor to apoenzymes while minimizing any toxic side reactions.

The C-3',4'-dideoxygenation structure in gentamicin can prevent deactivation by aminoglycoside 3'-phosphotransferase (APH(3')) in drug-resistant pathogens. However, the enzyme catalyzing the dideoxygenation step in the gentamicin biosynthesis pathway remains unknown.

Adaptive metabolic reprogramming gives cancer cells a proliferative advantage. Tumour cells extensively use glycolysis to sustain anabolism and produce serine, which not only refuels the one-carbon units necessary for the synthesis of nucleotide precursors and for DNA methylation, but also affects the cellular redox homeostasis. Given its central role in serine metabolism, serine hydroxymethyltransferase (SHMT), a pyridoxal 5'-phosphate (PLP)-dependent enzyme, is an attractive target for tumour chemotherapy. In humans, the cytosolic isoform (SHMT1) and the mitochondrial isoform (SHMT2) have distinct cellular roles, but high sequence identity and comparable catalytic properties, which may complicate development of successful therapeutic strategies. Here, we investigated how binding of the cofactor PLP controls the oligomeric state of the human isoforms. The fact that eukaryotic SHMTs are tetrameric proteins while bacterial SHMTs function as dimers may suggest that the quaternary assembly in eukaryotes provides an advantage to fine-tune SHMT function and differentially regulate intertwined metabolic fluxes, and may provide a tool to address the specificity problem. We determined the crystal structure of SHMT2, and compared it to the apo-enzyme structure, showing that PLP binding triggers a disorder-to-order transition accompanied by a large rigid-body movement of the two cofactor-binding domains. Moreover, we demonstrated that SHMT1 exists in solution as a tetramer, both in the absence and presence of PLP, while SHMT2 undergoes a dimer-to-tetramer transition upon PLP binding. These findings indicate an unexpected structural difference between the two human SHMT isoforms, which opens new perspectives for understanding their differing behaviours, roles or regulation mechanisms in response to PLP availability in vivo.