Pyridoxine 5’-phosphate oxidase is an enzyme

Enzyme

Enzymes are proteins that catalyze chemical reactions. In enzymatic reactions, the molecules at the beginning of the process, called substrates, are converted into different molecules, called products. Almost all chemical reactions in a biological cell need enzymes in order to occur at rates...

 that catalyzes several reactions in the vitamin B₆

Vitamin B6

Vitamin B6 is a water-soluble vitamin and is part of the vitamin B complex group. Several forms of the vitamin are known, but pyridoxal phosphate is the active form and is a cofactor in many reactions of amino acid metabolism, including transamination, deamination, and decarboxylation...

 metabolism pathway. Pyridoxine-5-P oxidase catalyzes the terminal step (also the rate-limiting step) in vitamin B₆ metabolism, the biosynthesis of pyridoxal-5’-phosphate, the biologically active form of vitamin B₆ which acts as an essential cofactor. Pyridoxine 5’-phosphate oxidase is a member of the enzyme class oxidase

Oxidase

An oxidase is any enzyme that catalyzes an oxidation-reduction reaction involving molecular oxygen as the electron acceptor. In these reactions, oxygen is reduced to water or hydrogen peroxide ....

s, or more specifically, oxidoreductases. These enzymes catalyze a simultaneous oxidation-reduction reaction. The substrate oxidase enzymes is hydroxlyated by one oxygen atom of molecular oxygen.
Concurrently, the other oxygen atom is reduced to water. Even though molecular oxygen is the electron acceptor in these enzymes’ reactions, they are unique because oxygen does not appear in the oxidized product.

Structure

Pyridoxine 5’-phosphate oxidase is a homodimer, or a molecule consisting of two identical polypeptide subunits. It is hypothesized that the two monomers are held together via disulfide bond

Disulfide bond

In chemistry, a disulfide bond is a covalent bond, usually derived by the coupling of two thiol groups. The linkage is also called an SS-bond or disulfide bridge. The overall connectivity is therefore R-S-S-R. The terminology is widely used in biochemistry...

s. There are also salt-bridge interactions between the two monomers. Each subunit tightly binds one molecule of pyridoxal 5'-phosphate tightly on each subunit. Both alpha-helices and beta-sheets are present in the protein motif, which is best described as a split-barrel structure. This structure is due, in part, to the disulfide bonds present in the secondary protein structure of this enzyme. Multiple thiol groups (-SH) indicate the presence of disulfide bonds in the structure of the molecule. This enzyme requires the presence of a cofactor, FMN (flavin mononucleotide

Flavin mononucleotide

Flavin mononucleotide , or riboflavin-5′-phosphate, is a biomolecule produced from riboflavin by the enzyme riboflavin kinase and functions as prosthetic group of various oxidoreductases including NADH dehydrogenase as well as cofactor in biological blue-light photo receptors...

).
Cofactors are ions or coenzymes necessary for enzyme activity. The FMN is located in a deep cleft (formed by the two polypeptide subunits), and held in place by extensive hydrogen-bond interactions with the protein. In this particular case, the FMN helps the enzyme to bind the substrates. In the absence of pyridoxal 5’-phosphate (PLP), the active site

Active site

In biology the active site is part of an enzyme where substrates bind and undergo a chemical reaction. The majority of enzymes are proteins but RNA enzymes called ribozymes also exist. The active site of an enzyme is usually found in a cleft or pocket that is lined by amino acid residues that...

 of the enzyme is in an “open” conformation. Once substrate binds and is converted to PLP, the active site of the enzyme is in a partially “closed” conformation. Specific amino acid residues can form hydrogen bonds with the PLP, thus forming a lid that physically covers the active site, giving rise to the “closed” conformation.

Pathway

Pyridoxine 5’-phosphate oxidase is the enzyme that catalyzes the rate-limited step of the B₆ metabolism pathway. Vitamin B₆, which is also known as pyridoxine

Pyridoxine

Pyridoxine is one of the compounds that can be called vitamin B6, along with pyridoxal and pyridoxamine. It differs from pyridoxamine by the substituent at the '4' position. It is often used as 'pyridoxine hydrochloride'.-Chemistry:...

, is a crucial nutrient for the human body, as it is responsible for more bodily functions that any other vitamin. Vitamin B₆ is a coenzyme in the metabolism of carbohydrates, fats and proteins. This means that the enzymes which break these entities down for use in the body cannot function unless Vitamin B₆ is present to induce a conformational change in the enzyme, thus activating it. Vitamin B₆ also plays a role in the synthesis of hormones, red blood cells, neurotransmitters and enzymes. A person who is deficient in Vitamin B₆ could suffer from insomnia, as well as suffer damage to the central nervous system.

Reactions

Pyridoxine 5’-phosphate oxidase catalyzes several reactions; the two most important are the deamination

Deamination

Deamination is the removal of an amine group from a molecule. Enzymes which catalyse this reaction are called deaminases.In the human body, deamination takes place primarily in the liver, however glutamate is also deaminated in the kidneys. Deamination is the process by which amino acids are...

 of pyridoxamine-5'-phosphate and the deamination of pyridoxine 5-phosphate, both of which are key intermediates in the metabolism of B₆. For a complete map of the B₆ metabolism, http://www.genome.ad.jp/dbget-bin/show_pathway?map00750+1.4.3.5 Pyridoxine 5’-phosphate oxidase’s EC number is 1.4.3.5.

pyridoxamine 5'-phosphate + H₂O + O₂ = pyridoxal 5'-phosphate + NH₃ + H₂O₂


pyridoxine phosphate + oxygen <=> H₂O₂ + pyridoxal phosphate


Pyridoxine 5’-phosphate oxidase also plays a role in nitrogen metabolism, converting amines to aldehydes NH₃ by the reaction: amine + H₂O + oxygen <=> aldehyde + NH₃ + H₂O₂

Kinetics

In humans, the pyridoxine 5’-phosphate oxidase enzyme exhibits a low catalytic rate constant of 0.2 sec-1, with low Km values for both pyridoxine 5'phosphate and pyridoxamine 5'-phosphate. The enzyme also has a low turnover rate, meaning that it is relatively slow converting substrate to product. Pyridoxal 5'-phosphate is an effective product inhibitor. Since pyridoxal 5'-phosphate, the active form of vitamin B₆, is the product of the metabolic pathway, if it exists in excess, then the pathway need not proceed to keep making product. However, if it exists in low concentrations, then that is a signal for the pathway to synthesize more. This is an example of feedback inhibition.

Pyridoxine 5’-phosphate oxidase in different organisms

Pyridoxine 5’-phosphate oxidase has been highly conserved over time, as there are many similarities between the enzyme as it is found in humans and E. coli. Although there is only 39% retention of amino acid sequence from the E. coli version of the enzyme to the human version, the sequences for the FMN binding site and the substrate active sites are among the very highly conserved portion. One of the key differences is that the human pyridoxine 5’-phosphate oxidase has a higher specificity for the pyridoxamine-5'-phosphate substrate, whereas the pyridoxine 5’-phosphate oxidase in E. coli has a higher specificity pyridoxal-5’-phosphate substrate.