Pyridoxine 5′-phosphate oxidase
Pyridoxine 5′-phosphate oxidase is an enzyme, encoded by the PNPO gene, that catalyzes several reactions in the vitamin B metabolism pathway. Pyridoxine 5′-phosphate oxidase catalyzes the final, rate-limiting step in vitamin B metabolism, the biosynthesis of pyridoxal 5′-phosphate, the biologically active form of vitamin B which acts as an essential cofactor. Pyridoxine 5′-phosphate oxidase is a member of the enzyme class oxidases, 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. The active form of vitamin B, pyridoxal 5'-phosphate (PLP), is critical for normal cellular function. Some cancer cells have notable differences in vitamin B metabolism compared to their normal counterparts. The rate-limiting enzyme in vitamin B synthesis is pyridoxine-5'-phosphate oxidase (PNPO; EC 1.4.3.5).[supplied by OMIM] 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 by disulfide bonds. 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). 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 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 B metabolism pathway. Vitamin B, which is also known as pyridoxine, is a crucial nutrient for the human body, as it is responsible for more bodily functions than any other vitamin. Vitamin B 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 B is present to induce a conformational change in the enzyme, thus activating it. Vitamin B also plays a  role in the synthesis of hormones, red blood cells, neurotransmitters and enzymes. A person who is deficient in Vitamin B 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 of pyridoxamine 5′-phosphate and the deamination of pyridoxine 5-phosphate, both of which are key intermediates in the metabolism of B. Pyridoxine 5′-phosphate oxidase's EC number is 1.4.3.5. Pyridoxine 5′-phosphate oxidase also plays a role in nitrogen metabolism, converting amines to aldehydes and NH by the reaction: Kinetics. In humans, the  pyridoxine 5′-phosphate oxidase enzyme exhibits a low catalytic rate constant of 0.2 s, with low "K" 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 B, 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 "Escherichia 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. Model organisms. Model organisms have been used in the study of PNPO function. A conditional knockout mouse line, called "Pnpo" was generated as part of the International Knockout Mouse Consortium program - a high-throughput mutagenesis project to generate and distribute animal models of disease to interested scientists. Male and female animals underwent a standardized phenotypic screen to determine the effects of deletion. Twenty four tests were carried out on mutant mice and two significant abnormalities were observed. No homozygous mutant embryos were identified during gestation, and  therefore none survived until weaning. The remaining tests were carried out on heterozygous mutant adult mice; no additional significant abnormalities were observed in these animals.