Vitamin B6 rescues insulin resistance and glucose-induced DNA damage caused by reduced activity of Drosophila PI3K.

The insulin signaling pathway controls cell growth and metabolism, thus its deregulation is associated with both cancer and diabetes. Phosphatidylinositol 3-kinase (PI3K) contributes to the cascade of phosphorylation events occurring in the insulin pathway by activating the protein kinase B (PKB/AKT), which phosphorylates several substrates, including those involved in glucose uptake and storage. PI3K inactivating mutations are associated with insulin resistance while activating mutations are identified in human cancers. Here we show that RNAi-induced depletion of the Drosophila PI3K catalytic subunit (Dp110) results in diabetic phenotypes such as hyperglycemia, body size reduction, and decreased glycogen content. Interestingly, we found that hyperglycemia produces chromosome aberrations (CABs) triggered by the accumulation of advanced glycation end-products and reactive oxygen species. Rearing PI3KRNAi flies in a medium supplemented with pyridoxal 5'-phosphate (PLP; the catalytically active form of vitamin B6) rescues DNA damage while, in contrast, treating PI3KRNAi larvae with the PLP inhibitor 4-deoxypyridoxine strongly enhances CAB frequency. Interestingly, PLP supplementation rescues also diabetic phenotypes. Taken together, our results provide a strong link between impaired PI3K activity and genomic instability, a crucial relationship that needs to be monitored not only in diabetes due to impaired insulin signaling but also in cancer therapies based on PI3K inhibitors. In addition, our findings confirm the notion that vitamin B6 is a good natural remedy to counteract insulin resistance and its complications.

Characterization of Novel Pathogenic Variants Causing Pyridox(am)ine 5'-Phosphate Oxidase-Dependent Epilepsy.

Several variants of the enzyme pyridox(am)ine 5'-phosphate oxidase (PNPO), responsible for a rare form of vitamin B6-dependent neonatal epileptic encephalopathy known as PNPO deficiency (PNPOD), have been reported. However, only a few of them have been characterised with respect to their structural and functional properties, despite the fact that the knowledge of how variants affect the enzyme may clarify the disease mechanism and improve treatment. Here, we report the characterisation of the catalytic, allosteric and structural properties of recombinantly expressed D33V, R161C, P213S, and E50K variants, among which D33V (present in approximately 10% of affected patients) is one of the more common variants responsible for PNPOD. The D33V and E50K variants have only mildly altered catalytic properties. In particular, the E50K variant, given that it has been found on the same chromosome with other known pathogenic variants, may be considered non-pathogenic. The P213S variant has lower thermal stability and reduced capability to bind the FMN cofactor. The variant involving Arg161 (R161C) largely decreases the affinity for the pyridoxine 5'-phosphate substrate and completely abolishes the allosteric feedback inhibition exerted by the pyridoxal 5'-phosphate product.

Physiological and Biochemical Markers of the Sex-Specific Sensitivity to Epileptogenic Factors, Delayed Consequences of Seizures and Their Response to Vitamins B1 and B6 in a Rat Model.

The disturbed metabolism of vitamins B1 or B6, which are essential for neurotransmitters homeostasis, may cause seizures. Our study aims at revealing therapeutic potential of vitamins B1 and B6 by estimating the short- and long-term effects of their combined administration with the seizure inductor pentylenetetrazole (PTZ). The PTZ dose dependence of a seizure and its parameters according to modified Racine's scale, along with delayed physiological and biochemical consequences the next day after the seizure are assessed regarding sexual dimorphism in epilepsy. PTZ sensitivity is stronger in the female than the male rats. The next day after a seizure, sex differences in behavior and brain biochemistry arise. The induced sex differences in anxiety and locomotor activity correspond to the disappearance of sex differences in the brain aspartate and alanine, with appearance of those in glutamate and glutamine. PTZ decreases the brain malate dehydrogenase activity and urea in the males and the phenylalanine in the females. The administration of vitamins B1 and B6 24 h before PTZ delays a seizure in female rats only. This desensitization is not observed at short intervals (0.5-2 h) between the administration of the vitamins and PTZ. With the increasing interval, the pyridoxal kinase (PLK) activity in the female brain decreases, suggesting that the PLK downregulation by vitamins contributes to the desensitization. The delayed effects of vitamins and/or PTZ are mostly sex-specific and interacting. Our findings on the sex differences in sensitivity to epileptogenic factors, action of vitamins B1/B6 and associated biochemical events have medical implications.

A Novel, Easy Assay Method for Human Cysteine Sulfinic Acid Decarboxylase.

Cysteine sulfinic acid decarboxylase catalyzes the last step of taurine biosynthesis in mammals, and belongs to the fold type I superfamily of pyridoxal-5'-phosphate (PLP)-dependent enzymes. Taurine (2-aminoethanesulfonic acid) is the most abundant free amino acid in animal tissues; it is highly present in liver, kidney, muscle, and brain, and plays numerous biological and physiological roles. Despite the importance of taurine in human health, human cysteine sulfinic acid decarboxylase has been poorly characterized at the biochemical level, although its three-dimensional structure has been solved. In the present work, we have recombinantly expressed and purified human cysteine sulfinic acid decarboxylase, and applied a simple spectroscopic direct method based on circular dichroism to measure its enzymatic activity. This method gives a significant advantage in terms of simplicity and reduction of execution time with respect to previously used assays, and will facilitate future studies on the catalytic mechanism of the enzyme. We determined the kinetic constants using L-cysteine sulfinic acid as substrate, and also showed that human cysteine sulfinic acid decarboxylase is capable to catalyze the decarboxylation-besides its natural substrates L-cysteine sulfinic acid and L-cysteic acid-of L-aspartate and L-glutamate, although with much lower efficiency.

Knowns and Unknowns of Vitamin B6 Metabolism in Escherichia coli.

Vitamin B6 is an ensemble of six interconvertible vitamers: pyridoxine (PN), pyridoxamine (PM), pyridoxal (PL), and their 5'-phosphate derivatives, PNP, PMP, and PLP. Pyridoxal 5'-phosphate is a coenzyme in a variety of enzyme reactions concerning transformations of amino and amino acid compounds. This review summarizes all known and putative PLP-binding proteins found in the Escherichia coli MG1655 proteome. PLP can have toxic effects since it contains a very reactive aldehyde group at its 4' position that easily forms aldimines with primary and secondary amines and reacts with thiols. Most PLP is bound either to the enzymes that use it as a cofactor or to PLP carrier proteins, protected from the cellular environment but at the same time readily transferable to PLP-dependent apoenzymes. E. coli and its relatives synthesize PLP through the seven-step deoxyxylulose-5-phosphate (DXP)-dependent pathway. Other bacteria synthesize PLP in a single step, through a so-called DXP-independent pathway. Although the DXP-dependent pathway was the first to be revealed, the discovery of the widespread DXP-independent pathway determined a decline of interest in E. coli vitamin B6 metabolism. In E. coli, as in most organisms, PLP can also be obtained from PL, PN, and PM, imported from the environment or recycled from protein turnover, via a salvage pathway. Our review deals with all aspects of vitamin B6 metabolism in E. coli, from transcriptional to posttranslational regulation. A critical interpretation of results is presented, in particular, concerning the most obscure aspects of PLP homeostasis and delivery to PLP-dependent enzymes.

Molecular characterization of pyridoxine 5'-phosphate oxidase and its pathogenic forms associated with neonatal epileptic encephalopathy.

Defects of vitamin B6 metabolism are responsible for severe neurological disorders, such as pyridoxamine 5'-phosphate oxidase deficiency (PNPOD; OMIM: 610090), an autosomal recessive inborn error of metabolism that usually manifests with neonatal-onset severe seizures and subsequent encephalopathy. At present, 27 pathogenic mutations of the gene encoding human PNPO are known, 13 of which are homozygous missense mutations; however, only 3 of them have been characterised with respect to the molecular and functional properties of the variant enzyme forms. Moreover, studies on wild type and variant human PNPOs have so far largely ignored the regulation properties of this enzyme. Here, we present a detailed characterisation of the inhibition mechanism of PNPO by pyridoxal 5'-phosphate (PLP), the reaction product of the enzyme. Our study reveals that human PNPO has an allosteric PLP binding site that plays a crucial role in the enzyme regulation and therefore in the regulation of vitamin B6 metabolism in humans. Furthermore, we have produced, recombinantly expressed and characterised several PNPO pathogenic variants responsible for PNPOD (G118R, R141C, R225H, R116Q/R225H, and X262Q). Such replacements mainly affect the catalytic activity of PNPO and binding of the enzyme substrate and FMN cofactor, leaving the allosteric properties unaltered.

Interaction of Bacillus subtilis GabR with the gabTD promoter: role of repeated sequences and effect of GABA in transcriptional activation.

Bacillus subtilis is able to use γ-aminobutyric acid (GABA) found in the soil as carbon and nitrogen source, through the action of GABA aminotransferase (GabT) and succinic semialdehyde dehydrogenase (GabD). GABA acts as molecular effector in the transcriptional activation of the gabTD operon by GabR. GabR is the most studied member of the MocR family of prokaryotic pyridoxal 5'-phosphate (PLP)-dependent transcriptional regulators, yet crucial aspects of its mechanism of action are unknown. GabR binds to the gabTD promoter, but transcription is activated only when GABA is present. Here, we demonstrated, in contrast with what had been previously proposed, that three repeated nucleotide sequences in the promoter region, two direct repeats and one inverted repeat, are specifically recognized by GabR. We carried out in vitro and in vivo experiments using mutant forms of the gabTD promoter. Our results showed that GABA activates transcription by changing the modality of interaction between GabR and the recognized sequence repeats. A hypothetical model is proposed in which GabR exists in two alternative conformations that, respectively, prevent or promote transcription. According to this model, in the absence of GABA, GabR binds to DNA interacting with all three sequence repeats, overlapping the RNA polymerase binding site and therefore preventing transcription activation. On the other hand, when GABA binds to GabR, a conformational change of the protein leads to the release of the interaction with the inverted repeat, allowing transcription initiation by RNA polymerase.

Isolation of a Complex Formed Between Acinetobacter baumannii HemA and HemL, Key Enzymes of Tetrapyrroles Biosynthesis.

Plants, algae and most bacteria synthesize 5-aminolevulinic acid (ALA), the universal precursor of tetrapyrroles such as heme, chlorophyll and coenzyme B12, by a two-step transformation involving the NADPH-dependent glutamyl-tRNA reductase (HemA), which reduces tRNA-bound glutamate to glutamate-1-semialdehyde (GSA), and the pyridoxamine 5'-phosphate-dependent glutamate-1-semialdehyde-2,1-aminomutase (HemL), responsible for the isomerization of GSA into ALA. Since GSA is a very unstable compound at pH values around neutrality, the formation of a HemA-HemL complex has been proposed to occur, allowing for direct channeling of this intermediate from HemA to HemL. Experimental evidence of the formation of this complex has been obtained with the enzymes from Escherichia coli and Chlamydomonas reinhardtii. However, its isolation has never been attained, probably because HemA is degraded when intracellular heme accumulates. In this work, we devised a co-expression and co-purification strategy of HemA and HemL from Acinetobacter baumannii, which allowed the isolation of the HemA-HemL complex. Our results indicate that HemA is stabilized when co-expressed with HemL. The addition of citrate throughout the expression and purification procedure further promotes the formation of the HemA-HemL complex, which can be isolated in fair amount for functional and structural studies. This work lays the bases for a rational design of HemA-HemA inhibitors to be developed as antibacterial agents against A. baumannii, a multidrug resistant opportunistic pathogen responsible for a broad range of severe nosocomial infections.

Human Cytosolic and Mitochondrial Serine Hydroxymethyltransferase Isoforms in Comparison: Full Kinetic Characterization and Substrate Inhibition Properties.

Serine hydroxymethyltransferase (SHMT) catalyzes the reversible conversion of l-serine and tetrahydrofolate into glycine and 5,10-methylenetetrahydrofolate. This enzyme, which plays a pivotal role in one-carbon metabolism, is involved in cancer metabolic reprogramming and is a recognized target of chemotherapy intervention. In humans, two isoforms of the enzyme exist, which are commonly termed cytosolic SHMT1 and mitochondrial SHMT2. Considerable attention has been paid to the structural, mechanistic, and metabolic features of these isozymes. On the other hand, a detailed comparison of their catalytic and regulatory properties is missing, although this aspect seems to be considerably important, considering that SHMT1 and SHMT2 reside in different cellular compartments, where they play distinct roles in folate metabolism. Here we performed a full kinetic characterization of the serine hydroxymethyltransferase reaction catalyzed by SHMT1 and SHMT2, with a focus on pH dependence and substrate inhibition. Our investigation, which allowed the determination of all kinetic parameters of serine hydroxymethyltransferase forward and backward reactions, uncovered a previously unobserved substrate inhibition by l-serine and highlighted several interesting differences between SHMT1 and SHMT2. In particular, SHMT2 maintains a pronounced tetrahydrofolate substrate inhibition even at the alkaline pH characteristic of the mitochondrial matrix, whereas with SHMT1 this is almost abolished. At this pH, SHMT2 also shows a catalytic efficiency that is much higher than that of SHMT1. These observations suggest that such different properties represent an adaptation of the isoforms to the respective cellular environments and that substrate inhibition may be a form of regulation.

The MocR-like transcription factors: pyridoxal 5'-phosphate-dependent regulators of bacterial metabolism.

Many biological functions played by current proteins were not created by evolution from scratch, rather they were obtained combining already available protein scaffolds. This is the case of MocR-like bacterial transcription factors (MocR-TFs), a subclass of GntR transcription regulators, whose structure is the outcome of the fusion between DNA-binding proteins and pyridoxal 5'-phosphate (PLP)-dependent enzymes. The resultant chimeras can count on the properties of both protein classes, i.e. the capability to recognize specific DNA sequences and to bind PLP and amino-compounds; it is the modulation of such binding properties to confer to MocR-TFs chimeras the ability to interact with effector molecules and DNA so as to regulate transcription. MocR-TFs control different metabolic processes involving vitamin B6 and amino acids, which are canonical ligands of PLP-dependent enzymes. However, MocR-TFs are also implicated in the metabolism of compounds that are not substrates of PLP-dependent enzymes, such as rhizopine and ectoine. Genomic analyses show that MocR-TFs are widespread among eubacteria, implying an essential role in their metabolism and highlighting the scarcity of our knowledge on these important players in microbial metabolism. Although MocR-TFs have been discovered 15 years ago, the research activity on these transcriptional regulators has only recently intensified, producing a wealth of information that needs to be brought back to general principles. This is the main task of this review, which reports and analyses the available information concerning MocR-TFs functional role, structural features, interaction with effector molecules and the characteristics of DNA transcriptional factor-binding sites of MocR-based regulatory systems.

Self-Directed in Cell Production of Methionine Analogue Azidohomoalanine by Synthetic Metabolism and Its Incorporation into Model Proteins.

Common protocols for the incorporation of noncanonical amino acids (ncAAs) into proteins require addition of the desired ncAA to the growth medium, its cellular uptake, and subsequent intracellular accumulation. This feeding scheme is generally suitable for small-scale proof-of-concept incorporation experiments. However, it is no general solution for orthogonal translation of ncAAs, as their chemical synthesis is generally tedious and expensive. Here, we describe a simple protocol that efficiently couples in situ semi-synthetic biosynthesis of L-azidohomoalanine and its incorporation into proteins at L-methionine (Met) positions. In our metabolically engineered Met-auxotrophic Escherichia coli strain, Aha is biosynthesized from externally added sodium azide and O-acetyl-L-homoserine as inexpensive precursors. This represents an efficient platform for expression of azide-containing proteins suitable for site-selective bioorthogonal strategies aimed at noninvasive protein modifications (Tornøe et al., J Org Chem 67:3057-3064, 2002; Kiick et al., Angew Chem Int Ed 39:2148-2152, 2000; Budisa, Angew Chem Int Ed Engl 47:6426-6463, 2004; van Hest, J Am Chem Soc 122:1282-1288, 2000).

Pyridoxine-5'-phosphate oxidase (Pnpo) deficiency: Clinical and biochemical alterations associated with the C.347g>A (P.·Arg116gln) mutation.

BACKGROUND: Pyridoxal-5'-phosphate oxidase (PNPO) deficiency presents as a severe neonatal encephalopathy responsive to pyridoxal-5'-phosphate (PLP) or pyridoxine. Recent studies widened the phenotype of this condition and detected genetic variants on PNPO gene whose pathogenic role and clinical expression remain to be established. OBJECTIVE: This paper aims to characterize the functional effects of the c.347G>A (p.Arg116Gln) mutation in the PNPO gene in order to define its pathogenicity and describe the clinical features of new patients with epilepsy carrying this mutation. METHODS: Arg116Gln protein variant was expressed as recombinant protein. The mutant protein was characterized with respect to structural and kinetic properties, thermal stability, binding constants of cofactor (FMN) and product (PLP). We also reviewed clinical data of 3 new patients carrying the mutation. RESULTS: The Arg116Gln mutation does not alter the overall enzyme structure and only slightly affects its catalytic efficiency; nevertheless, this mutation affects thermal stability of PNPO, reduces its affinity for FMN and impairs transfer of PLP to PLP-dependent enzymes. Three boys with seizure onset between 8months and 3years of age, carrying the Arg116Gln mutation, are described. These three patients exhibited different seizure types associated with interictal EEG abnormalities and slow background activity. Mild/moderate intellectual disability was observed in 2/3 patients. A dramatic therapeutic response to pyridoxine was observed in the only patient who still had active seizures when starting treatment, while in all three patients interictal EEG discharges and background activity improved after pyridoxine treatment was initiated. CONCLUSIONS: The reported data support a pathogenic role of the c.347G>A (p.Arg116Gln) mutation in PNPO deficiency. The later onset of symptoms and the milder epilepsy phenotype of these expand the disease phenotype.

Study of DNA binding and bending by Bacillus subtilis GabR, a PLP-dependent transcription factor.

BACKGROUND: GabR is a transcriptional regulator belonging to the MocR/GabR family, characterized by a N-terminal wHTH DNA-binding domain and a C-terminal effector binding and/or oligomerization domain, structurally homologous to aminotransferases (ATs). In the presence of γ-aminobutyrate (GABA) and pyridoxal 5'-phosphate (PLP), GabR activates the transcription of gabT and gabD genes involved in GABA metabolism. METHODS: Here we report a biochemical and atomic force microscopy characterization of Bacillus subtilis GabR in complex with DNA. Complexes were assembled in vitro to study their stoichiometry, stability and conformation. RESULTS: The fractional occupancy of the GabR cognate site suggests that GabR binds as a dimer with Kd of 10nM. Upon binding GabR bends the DNA by 80° as measured by anomalous electrophoretic mobility. With GABA we observed a decrease in affinity and conformational rearrangements compatible with a less compact nucleo-protein complex but no changes of the DNA bending angle. By employing promoter and GabR mutants we found that basic residues of the positively charged groove on the surface of the AT domain affect DNA affinity. CONCLUSIONS: The present data extend current understanding of the GabR-DNA interaction and the effect of GABA and PLP. A model for the GabR-DNA complex, corroborated by a docking simulation, is proposed. GENERAL SIGNIFICANCE: Characterization of the GabR DNA binding mode highlights the key role of DNA bending and interactions with bases outside the canonical direct repeats, and might be of general relevance for the action mechanism of MocR transcription factors.

Salmonella typhimurium PtsJ is a novel MocR-like transcriptional repressor involved in regulating the vitamin B6 salvage pathway.

The vitamin B6 salvage pathway, involving pyridoxine 5'-phosphate oxidase (PNPOx) and pyridoxal kinase (PLK), recycles B6 vitamers from nutrients and protein turnover to produce pyridoxal 5'-phosphate (PLP), the catalytically active form of the vitamin. Regulation of this pathway, widespread in living organisms including humans and many bacteria, is very important to vitamin B6 homeostasis but poorly understood. Although some information is available on the enzymatic regulation of PNPOx and PLK, little is known on their regulation at the transcriptional level. In the present work, we identified a new MocR-like regulator, PtsJ from Salmonella typhimurium, which controls the expression of the pdxK gene encoding one of the two PLKs expressed in this organism (PLK1). Analysis of pdxK expression in a ptsJ knockout strain demonstrated that PtsJ acts as a transcriptional repressor. This is the first case of a MocR-like regulator acting as repressor of its target gene. Expression and purification of PtsJ allowed a detailed characterisation of its effector and DNA-binding properties. PLP is the only B6 vitamer acting as effector molecule for PtsJ. A DNA-binding region composed of four repeated nucleotide sequences is responsible for binding of PtsJ to its target promoter. Analysis of binding stoichiometry revealed that protein subunits/DNA molar ratio varies from 4 : 1 to 2 : 1, depending on the presence or absence of PLP. Structural characteristics of DNA transcriptional factor-binding sites suggest that PtsJ binds DNA according to a different model with respect to other characterised members of the MocR subgroup.

Biochemical data from the characterization of a new pathogenic mutation of human pyridoxine-5'-phosphate oxidase (PNPO).

PNPO deficiency is responsible of severe neonatal encephalopathy, responsive to pyridoxal-5'-phosphate (PLP) or pyridoxine. Recent studies widened the phenotype of this condition and detected new genetic variants on PNPO gene, whose pathogenetic role and clinical expression remain to be established. One of these mutations, Arg116Gln, is of particular interest because of its later onset of symptoms (beyond the first months of life) and its peculiar epileptic manifestations in patients. This protein variant was expressed as recombinant protein in E coli, purified to homogeneity, and characterized with respect to structural and kinetic properties, stability, binding constants of cofactor flavin mononucleotide (FMN) and product (PLP) in order to define the molecular and structural bases of its pathogenicity. For interpretation and discussion of reported data, together with the description of clinical studies, refer to the article [1] (doi: 10.1016/j.ymgme.2017.08.003).

Molecular mechanism of PdxR ­ a transcriptional activator involved in the regulation of vitamin B6 biosynthesis in the probiotic bacterium Bacillus clausii.

Pyridoxal 5'-phosphate (PLP), the well-known active form of vitamin B6 , is an essential enzyme cofactor involved in a large number of metabolic processes. PLP levels need to be finely tuned in response to cell requirements; however, little is known about the regulation of PLP biosynthesis and recycling pathways. The transcriptional regulator PdxR activates transcription of the pdxST genes encoding PLP synthase. It is characterized by an N-terminal helix-turn-helix motif that binds DNA and an effector-binding C-terminal domain homologous to PLP-dependent enzymes. Although it is known that PLP acts as an anti-activator, the mechanism of action of PdxR is unknown. In the present study, we analyzed the biochemical and DNA-binding properties of PdxR from the probiotic Bacillus clausii. Spectroscopic measurements showed that PLP is the only B6 vitamer that acts as an effector molecule of PdxR. Binding of PLP to PdxR determines a protein conformational change, as detected by gel filtration chromatography and limited proteolysis experiments. We showed that two direct repeats and one inverted repeat are present in the DNA promoter region and PdxR is able to bind DNA fragments containing any combination of two of them. However, when PLP binds to PdxR, it modifies the DNA-binding properties of the protein, making it selective for inverted repeats. A molecular mechanism is proposed in which the two different DNA binding modalities of PdxR determined by the presence or absence of PLP are responsible for the control of pdxST transcription.

Molecular basis of E. coli L-threonine aldolase catalytic inactivation at low pH.

L-Threonine aldolases (TAs), a family of enzymes belonging to the fold-type I pyridoxal 5'-phosphate (PLP) dependent enzymes, play a role in catalyzing the reversible cleavage of l-3-hydroxy-α-amino acids to glycine and the corresponding aldehydes. Threonine aldolases have great biotechnological potential for the syntheses of pharmaceutically relevant drug molecules because of their stereospecificity. The pH-dependency of their catalytic activity, affecting reaction intermediates, led us to study the effect of low-pH on Escherichia coli TA (eTA) structure. We report here a low-pH crystal structure of eTA at 2.1 Å resolution, with a non-covalently bound uncleaved l-serine substrate, and a PLP cofactor bound as an internal aldimine. This structure contrasts with other eTA structures obtained at physiological pH that show products or substrates bound as PLP-external aldimines. The non-productive binding at low-pH is due to an unusual substrate serine binding orientation in which the α-amino group and carboxylate group are in the wrong positions (relative to the active site residues) as a result of protonation of the α-amino group of the serine, as well as the active site histidines, His83 and His126. Protonation of these residues prevents the characteristic nucleophilic attack of the α-amino group of substrate serine on C4' of PLP to form the external aldimine. Our study shows that at low pH the change in charge distribution at the active site can result in substrates binding in a non-productive orientation.

How pyridoxal 5'-phosphate differentially regulates human cytosolic and mitochondrial serine hydroxymethyltransferase oligomeric state.

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.