Orotate phosphoribosyltransferase

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Orotate phosphoribosyltransferase
2PSI - orotate phosphoribosyltransferase bound to substrates - S cerevisiae.png
S. cerevisiae orotate phosphoribosyl transferase bound to its substrates. From PDB file 2PS1. [1]
Identifiers
EC no. 2.4.2.10
CAS no. 9030-25-5
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Orotate phosphoribosyltransferase (OPRTase) or orotic acid phosphoribosyltransferase is an enzyme involved in pyrimidine biosynthesis. It catalyzes the formation of orotidine 5'-monophosphate (OMP) from orotate and phosphoribosyl pyrophosphate. [1] In yeast and bacteria, orotate phosphoribosyltransferase is an independent enzyme with a unique gene coding for the protein, whereas in mammals and other multicellular organisms, the catalytic function is carried out by a domain of the bifunctional enzyme UMP synthase (UMPS). [2]

Contents

Biological background

Reaction catalyzed by orotate phosphoribosyltransferase. Substrates PRPP and orotate are converted to orotidylate and inorganic pyrophosphate. Orotate phosphoribosyltransferase reaction.svg
Reaction catalyzed by orotate phosphoribosyltransferase. Substrates PRPP and orotate are converted to orotidylate and inorganic pyrophosphate.

As OPRTase is part of a bifunctional complex UMP synthase in humans, the function and stability of this enzyme is not necessarily directly associated with disorders in the human body. It is however reasonable to believe that a dysfunction in one of the enzymes will cause a dysfunction of the whole enzyme. [3] Defects in UMP synthase is associated with hypochromic anemia. [3] In mammals, this bifunctional enzyme UMPS converts orotic acid into uridine monophosphate (UMP). [4] Orotate phosphoribosyltransferase is located at the N-terminal domain of UMP synthase. This process happens in multiple steps with orotate phosphoribosyltransferase responsible for the first step of adding a ribose ring to orotate. [4] In this step, orotic acid is converted into orotidylate using PRPP (phosphoribosyl pyrophosphate) as a cosubstrate. This reaction is driven by the hydrolysis of pyrophosphate. [5] Orotidylate decarboxylase is located at the C-terminal domain of UMPS and converts this orotidylate intermediate into uridine monophosphate (also referred to as uridylate or UMP) via decarboxylation. [4] These two-steps are rapid and irreversible in mammals. In other pyrimidine auxotrophs that do not have this bifunctional enzyme, usually less complex organisms, two separate enzymes are required to carry out this reaction. [6] Both orotidylate and uridylate are major pyrimidine nucleotides, as uridylate is a precursor to RNA. Uridylate (UMP) is later converted to UDP via phosphorylation by UMP kinase and ATP and then nucleoside diphosphate kinase reversibly phosphorylates UDP to UTP. UTP can then be aminated through catalysis by cytidine triphosphate synthetase to from CTP. [5]

Enzyme mechanism

The reaction of orotic acid (orotate) to orotidylate is catalyzed by orotate phophoribosyltransferase with the cofactor PRPP, which is a cofactor commonly used for nucleotide synthesis. It transfers pyrophosphoryl groups very favorably with a ΔG of -8.3 + 0.5 kcal/mol. [6] Two main interactions attract PRPP to assist orotate phophoribosyltransferase in this reaction. First, orotate phophoribosyltransferase has Aspartic acid- Aspartic acid residues next to its PRPP-binding motif which interact with the ribosyl 2-/3- hydroxyl groups that stabilize the movement of Carbon-1 of the bound ribosyl group. [6] The stabilization occurs through a hydrogen bonding network of these hydroxyl groups with pyrophosphate, water and magnesium. [6] Second, the side-chains of the C-terminal end of the PRPP-binding motif interact favorably with PRPP’s 5-phosphate. [6]

Orotate phosphoribosyltransferase overall structure with orotic acid, magnesium and PRPP in the active site pocket. Orotate Phosphoribosyltransferase zoomed out.png
Orotate phosphoribosyltransferase overall structure with orotic acid, magnesium and PRPP in the active site pocket.

In B. subtilis, PRPP is bound to these two sites with a Kd of 33mM. When orotate is present, pyrophosphate binding affinity is increased fourfold and the reaction undergoes burst kinetics, with rapid phosphoribosyl transfer and then slow release of products. [7] This slow release is thought to be due to the solvent-exposed loop of orotate phosphoribosyltransferase that protects the active site during the first step. [7] The loop opening happens in two-steps with the PRPP dissociation unfavorable and slow since the loop closes 85% of the time. [8]

Three key pyrimidine nucleosides include uridine, cytidine and thymidine, and they play major roles in nucleic acid biosynthesis as well as carbohydrate and lipid metabolism. [4] Pyrimidine phosphoribosyltransferases such as orotate phosphoribosyltransferase activate their substrates by forming SN1-like transition states, facilitating migration of the ribosyl anomeric carbon region to MgPPi. [9] Like other pyrimidine phosphoribosyltransferases, orotate phosphoribosyltransferase has a flexible loop that moves to position groups in the ideal positions for catalysis. [4] They also use many water molecules to hold everything in place during the reaction. [6]

Enzyme structure

The crystal structure of OPRTase has been solved several times by various scientific groups. [1] [10] [11] [12] In bacteria, the overall structure is a dimer of two subunits, each consisting of seven α-helices and ten β-strands, with a molecular weight of 23919.13 Da. [13] Orotate phosphoribosyltransferase has a core part plus a flexible loop, which when closed prevents solvent from entering during reaction. [1] In other organisms such as mammals, insects and slime modes it is one of the domains of UMP synthase, with the other being orotidylate decarboxylase. [14] The N-terminal has a pair of antiparallel strands, with residues that interact with bound orotate and Lys 26 that extends to the active site and forms a bond with the flexible loop in its closed form. [6] Orotic acid and PRPP are stabilized in the active site mostly by hydrogen bonding with stabilizing interactions from Lys 26, Phe 34 and Phe 35 to orotic acid, as well as Thr 128, Ala 129, Gly 130, Ala 132, Asp 124, Lys 26 and Lys 73 to PRPP. [14] When Lys 26 is mutated, orotate phosphoribosyltransferase often exhibits reduced activity and specificity. [6]

Active site of orotate phosphoribosyltransferase bound to orotic acid and PRPP in Salmonella typhimurium. Stabilizing residues are shown, including Lys 26, Phe 34 and Phe 35 which stabilize orotic acid, as well as Thr 128, Ala 129, Gly 130, Ala 132, Asp 124, Lys 26 and Lys 73. Orotate Phosphoribosyltransferase.png
Active site of orotate phosphoribosyltransferase bound to orotic acid and PRPP in Salmonella typhimurium. Stabilizing residues are shown, including Lys 26, Phe 34 and Phe 35 which stabilize orotic acid, as well as Thr 128, Ala 129, Gly 130, Ala 132, Asp 124, Lys 26 and Lys 73.

Disease relevance

Defects in orotate phosphoribosyltransferase have been implicated in numerous medical conditions. Defects in the orotate phosphoribosyltransferase domain of UMPS cause orotic aciduria in humans, which is a rare hereditary metabolic disease resulting from problems with pyrimidine metabolism. [15] It can lead to megaloblastic anemia and orotic acid crystalluria, which is associated with physical and mental impairments. [15] Orotic aciduria was first reported in 1959 when excess orotic acid was found in the urine of an infant. [6] When individuals have a mutation leading to loss of orotate phosphoribosyltransferase activity and thus UTP production, orotic acid builds up and can be as high as 1.5g/day in infant urine. [6] This is because UTP is the normal end product in healthy individuals in the pyrimidine synthetic pathway and normally regulates the pathway. [6] Orotic acid buildup can lead to precipitation in the kidney and eventually renal failure. [6] Similarly, in Holstein cattle, UMPS deficiency is caused by an autosomal disorder which leads to death of offspring in the early embryonic state. [16]

Orotate phosphoribosyltransferase is also the main enzyme involved in converted 5-flurouracil to 5-F-UMP through phosphoribosylation. Some scientific studies have shown that orotate phosphoribosyltransferase potentially may play a role in cancer prognostics. [17] For instance, one study found that the ratio of gene expression of orotate phosphoribosyltransferase to dihydropyrimidine dehydrogenase affects the prognosis of metastatic colorectal cancer patients after fluropyrimidine-based chemotherapy. [17] 5-F-UMP is thought to become a suicide inhibitor for thymidylate synthetase and plays an important role in tumor growth inhibition. [17] [18] When resectable colorectal cancer patients were treated with oral 5-flurouracil, patients with high levels of orotate phosphoribosyltransferase had significantly better survival outcomes. [18] Similarly prognosis potential based on orotate phosphoribosyltransferase levels and activity have been implicated in bladder carcinoma and gastric carcinoma. [19] [20]

See also

Related Research Articles

<span class="mw-page-title-main">Nucleotide</span> Biological molecules constituting nucleic acids

Nucleotides are organic molecules composed of a nitrogenous base, a pentose sugar and a phosphate. They serve as monomeric units of the nucleic acid polymers – deoxyribonucleic acid (DNA) and ribonucleic acid (RNA), both of which are essential biomolecules within all life-forms on Earth. Nucleotides are obtained in the diet and are also synthesized from common nutrients by the liver.

<span class="mw-page-title-main">Uridine</span> One of the five major nucleosides in nucleic acids

Uridine (symbol U or Urd) is a glycosylated pyrimidine analog containing uracil attached to a ribose ring (or more specifically, a ribofuranose) via a β-N1-glycosidic bond. The analog is one of the five standard nucleosides which make up nucleic acids, the others being adenosine, thymidine, cytidine and guanosine. The five nucleosides are commonly abbreviated to their symbols, U, A, dT, C, and G, respectively. However, thymidine is more commonly written as 'dT' ('d' represents 'deoxy') as it contains a 2'-deoxyribofuranose moiety rather than the ribofuranose ring found in uridine. This is because thymidine is found in deoxyribonucleic acid (DNA) and usually not in ribonucleic acid (RNA). Conversely, uridine is found in RNA and not DNA. The remaining three nucleosides may be found in both RNA and DNA. In RNA, they would be represented as A, C and G whereas in DNA they would be represented as dA, dC and dG.

<span class="mw-page-title-main">Uridine monophosphate synthase</span> Protein-coding gene in the species Homo sapiens

The enzyme Uridine monophosphate synthase catalyses the formation of uridine monophosphate (UMP), an energy-carrying molecule in many important biosynthetic pathways. In humans, the gene that codes for this enzyme is located on the long arm of chromosome 3 (3q13).

<span class="mw-page-title-main">Ribonucleotide</span> Nucleotide containing ribose as its pentose component

In biochemistry, a ribonucleotide is a nucleotide containing ribose as its pentose component. It is considered a molecular precursor of nucleic acids. Nucleotides are the basic building blocks of DNA and RNA. Ribonucleotides themselves are basic monomeric building blocks for RNA. Deoxyribonucleotides, formed by reducing ribonucleotides with the enzyme ribonucleotide reductase (RNR), are essential building blocks for DNA. There are several differences between DNA deoxyribonucleotides and RNA ribonucleotides. Successive nucleotides are linked together via phosphodiester bonds.

A salvage pathway is a pathway in which a biological product is produced from intermediates in the degradative pathway of its own or a similar substance. The term often refers to nucleotide salvage in particular, in which nucleotides are synthesized from intermediates in their degradative pathway.

A nucleoside triphosphate is a nucleoside containing a nitrogenous base bound to a 5-carbon sugar, with three phosphate groups bound to the sugar. They are the molecular precursors of both DNA and RNA, which are chains of nucleotides made through the processes of DNA replication and transcription. Nucleoside triphosphates also serve as a source of energy for cellular reactions and are involved in signalling pathways.

In molecular biology, biosynthesis is a multi-step, enzyme-catalyzed process where substrates are converted into more complex products in living organisms. In biosynthesis, simple compounds are modified, converted into other compounds, or joined to form macromolecules. This process often consists of metabolic pathways. Some of these biosynthetic pathways are located within a single cellular organelle, while others involve enzymes that are located within multiple cellular organelles. Examples of these biosynthetic pathways include the production of lipid membrane components and nucleotides. Biosynthesis is usually synonymous with anabolism.

<span class="mw-page-title-main">Uridine monophosphate</span> Chemical compound

Uridine monophosphate (UMP), also known as 5′-uridylic acid, is a nucleotide that is used as a monomer in RNA. It is an ester of phosphoric acid with the nucleoside uridine. UMP consists of the phosphate group, the pentose sugar ribose, and the nucleobase uracil; hence, it is a ribonucleotide monophosphate. As a substituent or radical its name takes the form of the prefix uridylyl-. The deoxy form is abbreviated dUMP. Covalent attachment of UMP is called uridylylation.

<span class="mw-page-title-main">Orotic acid</span> Chemical compound synthesized in the body via a mitochondrial enzyme

Orotic acid is a pyrimidinedione and a carboxylic acid. Historically, it was believed to be part of the vitamin B complex and was called vitamin B13, but it is now known that it is not a vitamin.

<span class="mw-page-title-main">Adenine phosphoribosyltransferase</span> Mammalian protein found in Homo sapiens

Adenine phosphoribosyltransferase (APRTase) is an enzyme encoded by the APRT gene, found in humans on chromosome 16. It is part of the Type I PRTase family and is involved in the nucleotide salvage pathway, which provides an alternative to nucleotide biosynthesis de novo in humans and most other animals. In parasitic protozoa such as giardia, APRTase provides the sole mechanism by which AMP can be produced. APRTase deficiency contributes to the formation of kidney stones (urolithiasis) and to potential kidney failure.

<span class="mw-page-title-main">Phosphoribosyl pyrophosphate</span> Chemical compound

Phosphoribosyl pyrophosphate (PRPP) is a pentose phosphate. It is a biochemical intermediate in the formation of purine nucleotides via inosine-5-monophosphate, as well as in pyrimidine nucleotide formation. Hence it is a building block for DNA and RNA. The vitamins thiamine and cobalamin, and the amino acid tryptophan also contain fragments derived from PRPP. It is formed from ribose 5-phosphate (R5P) by the enzyme ribose-phosphate diphosphokinase:

<span class="mw-page-title-main">Nucleic acid metabolism</span> Process

Nucleic acid metabolism is a collective term that refers to the variety of chemical reactions by which nucleic acids are either synthesized or degraded. Nucleic acids are polymers made up of a variety of monomers called nucleotides. Nucleotide synthesis is an anabolic mechanism generally involving the chemical reaction of phosphate, pentose sugar, and a nitrogenous base. Degradation of nucleic acids is a catabolic reaction and the resulting parts of the nucleotides or nucleobases can be salvaged to recreate new nucleotides. Both synthesis and degradation reactions require multiple enzymes to facilitate the event. Defects or deficiencies in these enzymes can lead to a variety of diseases.

<span class="mw-page-title-main">Orotidine 5'-phosphate decarboxylase</span>

Orotidine 5′-phosphate decarboxylase or orotidylate decarboxylase is an enzyme involved in pyrimidine biosynthesis. It catalyzes the decarboxylation of orotidine monophosphate (OMP) to form uridine monophosphate (UMP). The function of this enzyme is essential to the de novo biosynthesis of the pyrimidine nucleotides uridine triphosphate, cytidine triphosphate, and thymidine triphosphate. OMP decarboxylase has been a frequent target for scientific investigation because of its demonstrated extreme catalytic efficiency and its usefulness as a selection marker for yeast strain engineering.

Pyrimidine biosynthesis occurs both in the body and through organic synthesis.

<span class="mw-page-title-main">Ribose 5-phosphate</span> Chemical compound

Ribose 5-phosphate (R5P) is both a product and an intermediate of the pentose phosphate pathway. The last step of the oxidative reactions in the pentose phosphate pathway is the production of ribulose 5-phosphate. Depending on the body's state, ribulose 5-phosphate can reversibly isomerize to ribose 5-phosphate. Ribulose 5-phosphate can alternatively undergo a series of isomerizations as well as transaldolations and transketolations that result in the production of other pentose phosphates as well as fructose 6-phosphate and glyceraldehyde 3-phosphate.

<span class="mw-page-title-main">Orotidine 5'-monophosphate</span> Chemical compound

Orotidine 5'-monophosphate (OMP), also known as orotidylic acid, is a pyrimidine nucleotide which is the last intermediate in the biosynthesis of uridine monophosphate. OMP is formed from orotate and phosphoribosyl pyrophosphate by the enzyme orotate phosphoribosyltransferase.

Purine metabolism refers to the metabolic pathways to synthesize and break down purines that are present in many organisms.

<span class="mw-page-title-main">Amidophosphoribosyltransferase</span> Mammalian protein found in Homo sapiens

Amidophosphoribosyltransferase (ATase), also known as glutamine phosphoribosylpyrophosphate amidotransferase (GPAT), is an enzyme responsible for catalyzing the conversion of 5-phosphoribosyl-1-pyrophosphate (PRPP) into 5-phosphoribosyl-1-amine (PRA), using the amine group from a glutamine side-chain. This is the committing step in de novo purine synthesis. In humans it is encoded by the PPAT gene. ATase is a member of the purine/pyrimidine phosphoribosyltransferase family.

<span class="mw-page-title-main">Ribose-phosphate diphosphokinase</span> Class of enzymes

Ribose-phosphate diphosphokinase is an enzyme that converts ribose 5-phosphate into phosphoribosyl pyrophosphate (PRPP). It is classified under EC 2.7.6.1.

In enzymology, an UMP kinase is an enzyme that catalyzes the chemical reaction

References

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