ピリドキサールリン酸
Pyridoxal phosphate/ja
 理想化された骨格式
| |
 | |
| 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 | |
| MeSH | Pyridoxal+Phosphate |
PubChem CID
|
|
| UNII | |
| |
| Properties | |
| C8H10NO6P | |
| Molar mass | 247.142 g/mol |
| Density | 1.638±0.06 g/cm3 |
| Melting point | 139 to 142 °C (282 to 288 °F; 412 to 415 K) |
| Acidity (pKa) | 1.56 |
| Pharmacology | |
| A11HA06 (WHO) | |
| Hazards | |
| Flash point | 296.0±32.9 °C |
ピリドキサールリン酸（PLP、ピリドキサール5'-リン酸、P5P）は、ビタミンB6の活性型であり、様々な酵素反応における補酵素である。国際生化学分子生物学連合は140以上のPLP依存性活性をカタログ化しており、分類された全活性の約4%に相当する。PLPの多用途性は、基質と共有結合した後、親電子触媒として作用し、様々なタイプのカルバニオン反応中間体を安定化させる能力から生じる。
補酵素としての役割
PLPはすべてのトランスアミノ化反応、および特定の脱炭酸、脱アミノ化、アミノ酸のラセミ化反応において補酵素として働く。 PLPのアルデヒド基は、アミノトランスフェラーゼ酵素の特定のリジン基のε-アミノ基とシッフ塩基結合（内部アルジミン）を形成する。アミノ酸基質のα-アミノ基は、活性部位リジン残基のε-アミノ基を転位脱離として知られるプロセスで置換する。結果として生じる外部アルジミンは、プロトン、二酸化炭素、アミノ酸側鎖を失ってキノノイド中間体となり、いくつかの反応経路で求核剤として働くことができる。
トランスアミノ化では、脱プロトン化後にキノノイド中間体が別の位置でプロトンを受け入れ、ケチミンとなる。得られたケチミンは加水分解され、アミノ基が複合体上に残る。さらに、PLPはペルオサミンやデソサミンのような珍しい糖に作用するアミノトランスフェラーゼ（またはトランスアミナーゼ）によっても利用される。これらの反応では、PLPはグルタミン酸と反応し、そのα-アミノ基をPLPに転移させてリン酸ピリドキサミン（PMP）を作る。 PMPはその後、窒素を糖に転移させ、アミノ糖を作る。
PLPは、セリンデヒドラターゼやGDP-4-ケト-6-デオキシマンノース-3-デヒドラターゼ（ColD）が行う反応など、様々なβ-脱離反応にも関与している。
また、ヘム合成における縮合反応でも活性を示す。
PLPは、レボドパからドーパミンへの変換に関与し、興奮性神経伝達物質であるグルタミン酸から抑制性神経伝達物質であるGABAへの変換を促進し、SAMを脱炭酸させてポリアミンの前駆体であるプロピルアミンを形成させる。
人体における役割
Pyridoxal phosphate has numerous roles in human body. A few examples below:
- Metabolism and biosynthesis of serotonin. Pyridoxal phosphate is a cofactor of aromatic L-amino acids decarboxylase. This allows for conversion of 5-hydroxytryptophan (5-HTP) into serotonine (5-HT). This reaction takes place in serotonergic neurons.
- Metabolism and biosynthesis of histamine. Pyridoxal phosphate is a cofactor of L-histidine decarboxylase. This allows for conversion of histidine into histamine. This reaction takes place in Golgi apparatus in mast cells and in basophils. Next, histamine is stored in granularity in mast cells as a complex with acid residues of heparin proteoglycan while in basophils as a complex with chondroitine sulfate.
- Metabolism and biosynthesis of GABA (γ-aminobutyric acid). Pyridoxal phosphate is a cofactor of glutamic acid decarboxylase (GAD). This allows for conversion of glutamate into GABA. Reaction takes place in cytoplasm of termination of GABA-ergic neurons, therefore vitamin B6 deficiency may cause epileptic seizures in children. Pyridoxal phosphate also participates in the oxidative deamination of GABA, where it is a cofactor of GABA aminotransferase.
- Metabolism of ornithine. Pyridoxal phosphate is a cofactor of ornithine carboxylase.
- Transamination. Pyridoxal phosphate takes part in decomposition and synthesis of amino acids, fats, and carbohydrates, and in the biosynthesis of hormones, neurotransmitters, and heme.
Non-classical examples of PLP
PLP is also found on glycogen phosphorylase in the liver, where it is used to break down glycogen in glycogenolysis when glucagon or epinephrine 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. Human Serine 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.
Catalytic mechanism
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.
Specificity
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). Consequently, specificity is dictated by how the enzymes bind their substrates. An additional role in specificity is played by the ease of protonation of the pyridine ring nitrogen.
PLP-enzymes
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.
- Fold Type I — aspartate aminotransferase family
- Fold Type II — tryptophan synthase family
- Fold Type III — alanine racemase family (TIM-barrel)
- Fold Type IV — D-amino acid aminotransferase family
- Fold Type V — glycogen phosphorylase family
Biosynthesis
From vitamers
Animals are auxotroph for this enzyme co-factor and require it or an intermediate to be supplemented, hence its classification as a vitamin B6, unlike MoCo or CoQ10 for example. PLP is synthesized from pyridoxal by the enzyme pyridoxal kinase, requiring one ATP molecule. PLP is metabolized in the liver.
Prototrophy
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 in Escherichia coli and Bacillus subtilis, respectively. Despite the disparity in the starting compounds and the different number of steps required, the two pathways possess many commonalities.
DXP-dependent biosynthesis
The DXP-dependent biosynthetic route requires several steps and a convergence of two branches, one producing 3-hydroxy-1-aminoacetone phosphate from erythrose 4-phosphate, while the other (single enzyme) producing deoxyxylulose 5-phosphate (DXP) from glyceraldehyde 3-phosphate (GAP) and pyruvate. The condensation product of 3-hydroxy-1-aminoacetone phosphate and deoxyxylulose 5-phosphate is pyridoxine 5'-phosphate. The condensation is catalyzed by PNP synthase, encoded by pdxJ, which creates PNP (pyridoxine 5' phosphate). The final enzyme is PNP 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 in E. coli by enzymes encoded by epd, pdxB, serC and pdxA. These share mechanistical similarities and homology with the three enzymes in serine biosynthesis (serA (homologue of pdxB), serC, serB — however, epd is a homologue of gap), which points towards a shared evolutionary origin of the two pathways. In several species there are two homologues of the E. coli serC gene, generally one in a ser operon (serC), and the other in a pdx operon, in which case it is called pdxF.
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) in E. coli. The serendipitous pathway was very inefficient, but was possible due to the promiscuous activity of various enzymes. It started with 3-phosphohydroxypyruvate (the product of the serA-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 substate for 4-PHT dehydrogenase (pdxA).
DXP-independent biosynthesis
The DXP-independent PLP-biosynthetic route consists of a step catalyzed by PLP-synthase, an enzyme composed of two subunits. PdxS catalyzes the condensation of ribulose 5-phosphate, glyceraldehyde-3-phosphate, and ammonia, this latter molecules is produced by PdxT which catalyzes the production of ammonia from glutamine. PdxS is a (β/α)8 barrel (also known as a TIM-barrel) that forms a dodecamer.
Abiotic synthesis
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 with prebiotic compounds, substances which serve as a food source for beneficial bacteria). In fact, heating NH3 and Glycolaldehyde spontaneously forms a variety of pyridines, including pyridoxal. Under certain conditions, PLP is formed from cyanoacetylene, diacetylene, carbon monoxide, hydrogen, water, and a phosphoric acid.
Inhibitors
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 is chloroalanine, which inhibits a large number of enzymes.
Examples of inhibitors:
- Levothyroxine In rats given only 10 µg of D, L-thyroxine daily for 15 days, liver cysteine desulfhydrase activity disappears and serine and threonine dehydrase and alanine glutamate transaminase activities decrease about 40%. Either in vivo feeding of pyridoxal-5-phosphate or in vitro addition of the coenzyme to the liver preparations restores full activity to all these enzymes, and the slight in vitro inhibition in the presence of 10−5 M thyroxine is also reversed by pyridoxal-5-phosphate.
- The inactive form pyridoxine competitively inhibits the active pyridoxal-5'-phosphate. Consequently, symptoms of vitamin B6 supplementation in the pyridoxine form can mimic those of vitamin B6 deficiency; an effect which perhaps might be avoided by supplementing with P5P instead.
- AlaP (alanine phosphonate) inhibits alanine racemases, but its lack of specificity has prompted further designs of ALR inhibitors.
- Gabaculine and Vigabatrin inhibit GABA aminotransferase
- Canaline and 5-fluoromethylornithine inhibit ornithine aminotransferase
- Amino-oxy SAM inhibits ACC synthase
Evolution
Pyridoxal-5-phosphate (vitamin B6)-dependent enzymes have multiple evolutionary origins. The overall B6 enzymes diverged into four independent evolutionary lines: α family (i.e. aspartate aminotransferase), β family (serine dehydratase),D-alanine aminotransferase family and the alanine racemase family. An example of the evolutionary similarity in the Beta family is seen in the mechanism. The β enzymes are all lyases and catalyze reactions where Cα and Cβ participate. Overall, in the PLP-dependent enzymes, the PLP in every case is covalently attached via an imine bond to the amino group in the active site.