Pyridoxal phosphate

Pyridoxal phosphate
IUPAC name [(4-formyl-5-hydroxy-6-methylpyridin-3-yl)methoxy]phosphonic acid
Other names Pyridoxal 5-phosphate, PAL-P, PLP, Vitamin B6 phosphate
Identifiers
CAS number	54-47-7 Y
PubChem	1051
MeSH	Pyridoxal+Phosphate
ChEMBL	CHEMBL82202 N
ATC code	A11HA06
Jmol-3D images	Image 1
SMILES CC1=NC=C(C(=C1O) C=O)COP(=O)(O)O
Properties
Molecular formula	C8H10NO6P
Molar mass	247.142 g/mol
Density	1.638±0.06 g/cm3
Melting point	139-142°C
Acidity (pKa)	1.56
Hazards
Flash point	296.0±32.9 °C
N (verify) (what is: Y/N?) Except where noted otherwise, data are given for materials in their standard state (at 25 °C, 100 kPa)
Infobox references

Pyridoxal-phosphate (PLP, pyridoxal-5'-phosphate, P5P) is a prosthetic group of some enzymes. It is the active form of vitamin B₆, which comprises three natural organic compounds, pyridoxal, pyridoxamine and pyridoxine. This cofactor acts as an electron sink in order to stabilize carbanionic intermediates in both substitution and elimination reactions involving aminated compounds.

Role as a coenzyme

PLP acts as a coenzyme in all transamination reactions, and in some decarboxylation, deamination, and racemization reactions of amino acids. The aldehyde group of PLP forms a Schiff-base linkage (internal aldimine) with the ε-amino group of a specific lysine group of the aminotransferase 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 quinoid intermediate, which in turn can act as a nucleophile in several reaction pathways.

In transamination, after deprotonation the quinoid intermediate accepts a proton at a different position to become a ketimine. The resulting ketimine is hydrolysed so that the amino group remains on the complex. In addition, PLP is used by aminotransferases (or transaminases) that act upon unusual sugars such as perosamine and desosamine. In these reactions, the PLP reacts with glutamate, which transfers its alpha-amino group to PLP to make pyridoxamine phosphate (PMP). PMP then transfers its nitrogen to the sugar, making an amino sugar.

PLP is also involved in various beta-elimination reactions such as the reactions carried out by serine dehydratase and GDP-4-keto-6-deoxymannose-3-dehydratase (ColD).

It is also active in the condensation reaction in heme synthesis.

PLP plays a role in the conversion of levodopa into dopamine, allows the conversion of the excitatory neurotransmitter glutamate to the inhibitory neurotransmitter GABA, and allows SAM to be decarboxylated to form propylamine, which is a precursor to polyamines.

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 an active site histidine. In such a case, the histidine cannot form the internal aldimine, and, therefore, the cofactor never becomes covalently tethered to the enzyme. GDP-4-keto-6-deoxymannose-3-dehydratase (ColD) is an example of such an enzyme.

Catalytic Mechanism

The pyridoxal-5′-phosphate-dependent enzymes (PLP enzymes) catalyze a myriad of biochemical reactions. Although the scope of PLP-catalyzed reactions initially appears to be immensely diverse, there is a simple unifying principle: In the resting state, the cofactor (PLP) is covalently bonded to the amino group of an active site lysine, forming an internal aldimine. Once the amino substrate interacts with the active site, a new Schiff base is generated, commonly referred to as the external aldimine. Only after this step, the mechanistic pathway for each PLP-catalyzed reaction diverges. Density functional methods have been applied to investigate the transimination reaction, and the results have shown that the reaction involves three sequential steps: (i) formation of a tetrahedral intermediate with the active site lysine and the amino substrate bonded to the PLP cofactor; (ii) nondirect proton transfer between the amino substrate and the lysine residue; and (iii) formation of the external aldimine after the dissociation of the lysine residue. The overall reaction is exothermic (−12.0 kcal/mol), and the rate-limiting step is the second one with 12.6 kcal/mol for the activation energy

Mechanistic examples: racemization of alanine and lyation of cysteine.

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 IV — D-amino acid aminotransferase family
• Fold Type III — alanine racemase family (TIM-barrel)
• Fold Type V — glycogen phosphorylase family

Biosynthesis

From vitamers

Animals are auxotrophs for this enzyme cofactor 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. It 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, and have been studied extensively in Escherichia coli and Bacillus subtilis, respectively. Despite the disparity in number of steps required between the two pathways and in starting compounds, 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 condesation product of 3-hydroxy-1-aminoacetone phosphate and deoxyxylulose 5-phosphate is pyridoxine 5'-phosphate. The condensation is catalysed by PNP synthase, encoded by pdxJ, which creates PNP (pyridoxine 5' phosphate). The final enzyme is PNP oxidase (pdxH) which oxidises the 4' hydroxyl group to an aldehyde using dioxigen and releasing hydrogen peroxide.

The first branch is catalysed 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 &emdash;however, epd is a homologue of gap), which points towards a shared evolutonary 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), while the other in a pdx operon, in which case it is called pdxF.

A "serependipidous 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 serependipidous pathway was very inefficient, but was possible thanks 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 dephophorylated, resulting in an unstable intermediate that spontaneously decarboxylates (hence the presence of the phosphate in the serine biosynthetic pathway) to glycaldehyde. Glycaldehyde was condensed with glycine and the phosphoryated product was 4-phosphohydroxythreonine (4PHT), the canonical substate for 4PHT dehydrogenase (pdxA).

DXP-independent biosynthesis

The DXP-independent PLP-biosynthetic route consists of a step catalysed 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.

Prebiotic synthesis

The widespread distribution in central metabolism, especially amino acid biosynthesis, and its activity in the absence of enzymes suggests PLP may be a prebiotic compound. In fact, heating NH3 and glycoaldehyde spontaneous forms a variety of pyridines, including pyridoxal. Under certain conditions, cyanoacetylene, diacetylene, carbon monoxide, hydrogen, water, and a phosphoric acid form PLP.

Inhibitors

Due to the importance of PLP enzymes, several inhbitors are known.

One type of inhibitors form an electrophile with the PLP that irreversibly reacts with the active site lysine. Generally these are acetylenic compounds (e.g. propargylglycine) and vinylic compounds (e.g. vinylglycine). Another type of inhibitor inactivates the PLP, such as α-methyl or amino-oxy substrate analogs (e.g. α-methylglutamate). A further type of inhibitors have with good leaving groups that nucleophilically attack the PLP, such as chloroalanine, which inhibits a large number of enzymes.

Some examples of inhibitors:

• AlaP (alanine phosphonate) inihibits 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

Etymology

Pyridine

PLP stands for pyridoxal 5' phosphate and PMP stands for pyridoxamine 5' phosphate. Consequently, the L in PLP does not indicate a levorotatory chiral centre.

The common names pyridoxamine (aminomethyl group at position 4), pyroxidal (carbaldehyde group) and pyridoxine (alkaloid, has hydroxymethyl) derive from pyridine + oxy + a IUPAC suffix to distinguish them, pyridine, in turn, comes from the Greek πῦρ, πυρός (pyr, fire) + -ide + -ine. It should be noted that the oxy infix in pyridoxine is not based on Hantzsch–Widman nomenclature (the heterocyclic ring is simply a modified pyridine) and the pyr- root does not idicate it to be a dimeric acid anhydride (unlike pyrophosphate).

See also

• Aromatic-L-amino-acid decarboxylase
• Ornithine decarboxylase

External links

• A11HA06