 Idealisedskeletal formula | |
 Ball-and-stick model based on thecrystal structure.[1][2] Note that the phosphate andpyridine groups have reacted to form azwitterion and thealdehyde group ishydrated. | |
| Names | |
|---|---|
| Preferred IUPAC name (4-Formyl-5-hydroxy-6-methylpyridin-3-yl)methyl dihydrogen phosphate | |
| Other names Pyridoxal 5-phosphate, PAL-P, PLP, Vitamin B6 phosphate | |
| Identifiers | |
| |
3D model (JSmol) | |
| ChEBI | |
| ChEMBL | |
| ChemSpider | |
| DrugBank | |
| ECHA InfoCard | 100.000.190 |
| EC Number |
|
| KEGG | |
| MeSH | Pyridoxal+Phosphate |
| UNII | |
| |
| |
| Properties | |
| C8H10NO6P | |
| Molar mass | 247.142 g/mol |
| Density | 1.638±0.06 g/cm3[3] |
| Melting point | 139 to 142 °C (282 to 288 °F; 412 to 415 K)[4] |
| Acidity (pKa) | 1.56[3] |
| Pharmacology | |
| A11HA06 (WHO) | |
| Hazards | |
| Flash point | 296.0±32.9 °C[3] |
Except where otherwise noted, data are given for materials in theirstandard state (at 25 °C [77 °F], 100 kPa). | |
Pyridoxal phosphate (PLP,pyridoxal 5'-phosphate,P5P), the active form ofvitamin B6, is acoenzyme in a variety ofenzymatic reactions. TheInternational Union of Biochemistry and Molecular Biology has catalogued more than 140 PLP-dependent activities, corresponding to ~4% of all classified activities.[5] The versatility of PLP arises from its ability to covalently bind the substrate, and then to act as an electrophilic catalyst, thereby stabilizing different types of carbanionic reaction intermediates.
PLP acts as a coenzyme in alltransamination reactions, and in certaindecarboxylation,deamination, andracemization reactions ofamino acids.[6] The aldehyde group of PLP forms aSchiff-base linkage (internalaldimine) with the ε-amino group of a specific lysine group of theaminotransferase enzyme. The α-amino group of the amino acid substrate displaces the ε-amino group of the active-site lysine residue in a process known as transaldimination. The resulting external aldimine can lose a proton, carbon dioxide, or an amino acid sidechain to become a quinonoid intermediate, which in turn can act as a nucleophile in several reaction pathways.
In transamination, after deprotonation the quinonoid intermediate accepts a proton at a different position to become aketimine. The resulting ketimine is hydrolysed so that the amino group remains on the complex.[7] In addition, PLP is used by aminotransferases (or transaminases) that act upon unusual sugars such asperosamine anddesosamine.[8] In these reactions, the PLP reacts withglutamate, which transfers its alpha-amino group to PLP to make pyridoxamine phosphate (PMP). PMP then transfers its nitrogen to the sugar, making anamino sugar.
PLP is also involved in various beta-elimination reactions such as the reactions carried out byserine dehydratase andGDP-4-keto-6-deoxymannose-3-dehydratase (ColD).[8]
It is also active in the condensation reaction inheme synthesis.
PLP plays a role in the conversion oflevodopa intodopamine, facilitates the conversion of the excitatory neurotransmitter glutamate to the inhibitory neurotransmitterGABA, and allowsSAM to be decarboxylated to formpropylamine, which is a precursor to polyamines.
Pyridoxal phosphate has numerous roles in human body. A few examples below:
In normal conditions, the human intestine absorbs mainly nonphosphorylated B6 vitamers. The phosphorylated forms of B6 and the glucoside of pyridoxine can be hydrolyzed by intestinal phosphatases and an intestinal glycosidase, respectively, to promote passive diffusion of pyridoxamine, pyridoxine and pyridoxal. In the liver or intestine, they are then rephosphorylated by pyridoxal kinase (EC 2.7.1.35) to avoid inverse diffusion[13] Atransport protein / membrane carrier of PLP (and other phosphorylated forms of B6) is the human membrane enzyme NAPE-PLDN-acyl phosphatidylethanolamine-specific phospholipase D of theendocannabinoid system.[14] In the presence ofbile acids (e.g.,digestion), NAPE-PLD with its internal channel creates membrane-pores as dynamic conductive pathways through which the charged cofactors of vitamin B6 can diffuse through cell membranes and membranes of subcellular compartments (e.g., mitochondria, peroxisome, and endosome), where they exert the specific enzymatic activities. NAPE-PLD is thus functional to the intracellular uptake and mobilization of PLP, and to the increased demand of the cofactor in pathological conditions having an higher endocannabinoid tone. Intracellular-free PLP concentrations are maintained at approximately 1 μM to prevent inappropriate reactions. Proteins that bind PLP and help maintain low-free PLP concentrations include glycogen phosphorylase in muscle, hemoglobin in erythrocytes, albumin in plasma, and NAPE-PLD mainly in the brain, gut, liver, kidney and reproductive system. When B6 vitamers intake exceeds requirements, PLP is dephosphorylated (mainly in the liver) and the pyridoxal is oxidized to pyridoxic acid prior to excretion in urine.
PLP is also found onglycogen phosphorylase in the liver, where it is used to break downglycogen inglycogenolysis whenglucagon orepinephrine signals it to do so. However, this enzyme does not exploit the reactive aldehyde group, but instead utilizes the phosphate group on PLP to perform its reaction.
Although the vast majority of PLP-dependent enzymes form an internal aldimine with PLP via an active site lysine residue, some PLP-dependent enzymes do not have this lysine residue, but instead have a histidine in the active site. In such a case, the histidine cannot form the internal aldimine, and, therefore, the co-factor does not become covalently tethered to the enzyme.GDP-4-keto-6-deoxymannose-3-dehydratase (ColD) is an example of such an enzyme.[15]HumanSerine hydroxymethyltransferase 2 regulates one-carbon transfer reactions required for amino acid and nucleotide metabolism, and exists in dimeric and tetrameric forms. The dimeric SHMT2 variant is a potent inhibitor of the BRISC deubiquitylase enzyme complex, which regulates immune-based cell signaling. Recent studies show that SJMT2 tetramerization is induced by PLP. This prevents interaction with the BRISC deubiqutylase complex, potentially linking vitamin B6 levels and metabolism to inflammation.[16]
The pyridoxal-5′-phosphate-dependent enzymes (PLP enzymes) catalyze myriad reactions. Although the scope of PLP-catalyzed reactions appears to be immense, the unifying principle is the formation of an internal lysine-derived aldimine. Once the amino substrate interacts with the active site, a new Schiff base is generated, commonly referred to as the external aldimine. After this step, the pathway for each PLP-catalyzed reactions diverge.[17]
Specificity is conferred by the fact that, of the four bonds of the alpha-carbon of the amino acid aldimine state, the bond perpendicular to the pyridine ring will be broken (Dunathan Stereoelectronic Hypothesis).[18][19] Consequently, specificity is dictated by how the enzymes bind their substrates.An additional role in specificity is played by the ease of protonation of thepyridine ring nitrogen.[20]
PLP is retained in the active site not only thanks to the lysine, but also thanks to the interaction of the phosphate group and a phosphate binding pocket and to a lesser extent thanks to base stacking of the pyridine ring with an overhanging aromatic residue, generally tyrosine (which may also partake in the acid–base catalysis). Despite the limited requirements for a PLP binding pocket, PLP enzymes belong to only five different families. These families do not correlate well with a particular type of reaction. The five families are classified as fold types followed by a Roman numeral.[18]
Animals areauxotroph for this enzyme co-factor and require it or an intermediate to be supplemented, hence its classification as a vitamin, unlikeMoCo orCoQ10 for example.PLP is synthesized from pyridoxal by the enzymepyridoxal kinase (genePDXK), requiring one ATP molecule. PDXK can also produce 5'-phosphates of the other vitamers (pyridoxine andpyridoxamine),[21] withpyridoxine 5′-phosphate oxidase converting these alternative 5'-phosphates into PLP.[22]
PLP is made and metabolized in the liver. PLP and other vitamin B6 phosphates can be broken down bypyridoxal phosphatase, which removes the phosphate group.[22]
Two natural pathways for PLP are currently known: one requires deoxyxylulose 5-phosphate (DXP), while the other does not, hence they are known as DXP-dependent and DXP-independent. These pathways have been studied extensively inEscherichia coli andBacillus subtilis, respectively. Despite the disparity in the starting compounds and the different number of steps required, the two pathways possess many commonalities.[23]
The DXP-dependent biosynthetic route requires several steps and a convergence of two branches, one producing3-hydroxy-1-aminoacetone phosphate fromerythrose 4-phosphate, while the other (single enzyme) producingdeoxyxylulose 5-phosphate (DXP) fromglyceraldehyde 3-phosphate (GAP) andpyruvate. The condensation product of3-hydroxy-1-aminoacetone phosphate anddeoxyxylulose 5-phosphate ispyridoxine 5'-phosphate. The condensation is catalyzed byPNP synthase, encoded bypdxJ, which creates PNP (pyridoxine 5' phosphate).[24] The final enzyme isPNP oxidase (pdxH), which catalyzes the oxidation of the 4' hydroxyl group to an aldehyde using dioxigen, resulting in hydrogen peroxide.
The first branch is catalyzed inE. coli by enzymes encoded byepd,pdxB,serC andpdxA. These share mechanistical similarities and homology with the three enzymes in serine biosynthesis (serA (homologue ofpdxB),serC,serB — however,epd is a homologue ofgap), which points towards a shared evolutionary origin of the two pathways.[25] In several species there are two homologues of theE. coliserC gene, generally one in a ser operon (serC), and the other in a pdx operon, in which case it is calledpdxF.
A "serendipitous pathway" was found in an overexpression library that could suppress the auxotrophy caused by the deletion of pdxB (encoding erythronate 4 phosphate dehydrogenase) inE. coli. The serendipitous pathway was very inefficient, but was possible due to thepromiscuous activity of various enzymes. It started with 3-phosphohydroxypyruvate (the product of theserA-encoded enzyme in serine biosynthesis) and did not require erythronate-4-phosphate. 3PHP was dephosphorylated, resulting in an unstable intermediate that decarboxylates spontaneously (hence the presence of the phosphate in the serine biosynthetic pathway) to glycaldehyde. Glycaldehyde was condensed with glycine and the phosphorylated product was 4-phosphohydroxythreonine (4PHT), the canonical substrate for 4-PHT dehydrogenase (pdxA).[26]
The DXP-independent PLP-biosynthetic route consists of a step catalyzed byPyridoxal 5'-phosphate synthase (glutamine hydrolyzing), an enzyme composed of two subunits. PdxS catalyzes the condensation of ribulose 5-phosphate, glyceraldehyde-3-phosphate, andammonia, this latter molecules is produced by PdxT which catalyzes the production of ammonia fromglutamine. PdxS is a (β/α)8 barrel (also known as a TIM-barrel) that forms a dodecamer.[27]
The widespread utilization of PLP in central metabolism, especially in amino acid biosynthesis, and its activity in the absence of enzymes, suggests PLP may be a "prebiotic" compound—that is, one that predates the origin of organic life (not to be confused withprebiotic compounds, substances which serve as a food source for beneficial bacteria).[28]In fact, heatingNH3 andGlycolaldehyde spontaneously forms a variety of pyridines, including pyridoxal.[28] Under certain conditions, PLP is formed from cyanoacetylene, diacetylene, carbon monoxide, hydrogen, water, and a phosphoric acid.[29]
Several inhibitors of PLP enzymes are known.
One type of inhibitor forms an electrophile with PLP, causing it to irreversibly react with the active site lysine. Acetylenic compounds (e.g. propargylglycine) and vinylic compounds (e.g. vinylglycine) are such inhibitors.A different type of inhibitor inactivates PLP, and such are α-methyl and amino-oxy substrate analogs (e.g. α-methylglutamate). Still other inhibitors have good leaving groups that nucleophilically attack the PLP. Such ischloroalanine, which inhibits a large number of enzymes.[18]
Examples of inhibitors:
Pyridoxal-5-phosphate (vitamin B6)-dependent enzymes have multiple evolutionary origins. The overallB6 enzymes diverged into four independent evolutionary lines: α family (i.e.aspartate aminotransferase), β family (serine dehydratase), D-alanine aminotransferase family and thealanine racemase family. An example of the evolutionary similarity in the Beta family is seen in the mechanism. The β enzymes are alllyases and catalyze reactions where Cα and Cβ participate. Overall, in thePLP-dependent enzymes, the PLP in every case is covalently attached via an imine bond to the amino group in the active site.[34]
