OPRTase In Complex With OMP
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Orotate phosphoribosyltransferase (OPRTase) or orotic acid phosphoribosyltransferase is an
enzyme An enzyme () is a protein that acts as a biological catalyst by accelerating chemical reactions. The molecules upon which enzymes may act are called substrate (chemistry), substrates, and the enzyme converts the substrates into different mol ...
involved in
pyrimidine Pyrimidine (; ) is an aromatic, heterocyclic, organic compound similar to pyridine (). One of the three diazines (six-membered heterocyclics with two nitrogen atoms in the ring), it has nitrogen atoms at positions 1 and 3 in the ring. The oth ...
biosynthesis Biosynthesis, i.e., chemical synthesis occurring in biological contexts, is a term most often referring to multi-step, enzyme-Catalysis, catalyzed processes where chemical substances absorbed as nutrients (or previously converted through biosynthe ...
. It catalyzes the formation of orotidine 5'-monophosphate (OMP) from
orotate 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. The compound is synthesized in the body via ...
and
phosphoribosyl pyrophosphate Phosphoribosyl pyrophosphate (PRPP) is a Sugar phosphates, pentose phosphate. It is a biochemical intermediate in the formation of purine nucleotides via Inosinic acid, inosine-5-monophosphate, as well as in pyrimidine nucleotide formation. Hence ...
. In
yeast Yeasts are eukaryotic, single-celled microorganisms classified as members of the fungus kingdom (biology), kingdom. The first yeast originated hundreds of millions of years ago, and at least 1,500 species are currently recognized. They are est ...
and
bacteria Bacteria (; : bacterium) are ubiquitous, mostly free-living organisms often consisting of one Cell (biology), biological cell. They constitute a large domain (biology), domain of Prokaryote, prokaryotic microorganisms. Typically a few micr ...
, orotate phosphoribosyltransferase is an independent enzyme with a unique
gene In biology, the word gene has two meanings. The Mendelian gene is a basic unit of heredity. The molecular gene is a sequence of nucleotides in DNA that is transcribed to produce a functional RNA. There are two types of molecular genes: protei ...
coding for the protein, whereas in
mammals A mammal () is a vertebrate animal of the class Mammalia (). Mammals are characterised by the presence of milk-producing mammary glands for feeding their young, a broad neocortex region of the brain, fur or hair, and three middle e ...
and other multicellular
organisms An organism is any living thing that functions as an individual. Such a definition raises more problems than it solves, not least because the concept of an individual is also difficult. Many criteria, few of them widely accepted, have been pr ...
, the catalytic function is carried out by a domain of the bifunctional enzyme
UMP synthase The enzyme Uridine monophosphate synthase (, UMPS) (orotate phosphoribosyl transferase and orotidine-5'-decarboxylase) catalyses the formation of uridine monophosphate (UMP), an energy-carrying molecule in many important biosynthetic pathways. In ...
(UMPS).


Biological background

As OPRTase is part of a bifunctional complex
UMP synthase The enzyme Uridine monophosphate synthase (, UMPS) (orotate phosphoribosyl transferase and orotidine-5'-decarboxylase) catalyses the formation of uridine monophosphate (UMP), an energy-carrying molecule in many important biosynthetic pathways. In ...
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. Defects in
UMP synthase The enzyme Uridine monophosphate synthase (, UMPS) (orotate phosphoribosyl transferase and orotidine-5'-decarboxylase) catalyses the formation of uridine monophosphate (UMP), an energy-carrying molecule in many important biosynthetic pathways. In ...
is associated with hypochromic anemia. In mammals, this bifunctional enzyme UMPS converts
orotic acid 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. The compound is synthesized in the body via ...
into
uridine monophosphate Uridine monophosphate (UMP), also known as 5′-uridylic acid ( conjugate base uridylate), 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, th ...
(UMP). Orotate phosphoribosyltransferase is located at the N-terminal domain of
UMP synthase The enzyme Uridine monophosphate synthase (, UMPS) (orotate phosphoribosyl transferase and orotidine-5'-decarboxylase) catalyses the formation of uridine monophosphate (UMP), an energy-carrying molecule in many important biosynthetic pathways. In ...
. This process happens in multiple steps with orotate phosphoribosyltransferase responsible for the first step of adding a
ribose Ribose is a simple sugar and carbohydrate with molecular formula C5H10O5 and the linear-form composition H−(C=O)−(CHOH)4−H. The naturally occurring form, , is a component of the ribonucleotides from which RNA is built, and so this comp ...
ring to
orotate 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. The compound is synthesized in the body via ...
. In this step,
orotic acid 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. The compound is synthesized in the body via ...
is converted into orotidylate using PRPP (
phosphoribosyl pyrophosphate Phosphoribosyl pyrophosphate (PRPP) is a Sugar phosphates, pentose phosphate. It is a biochemical intermediate in the formation of purine nucleotides via Inosinic acid, inosine-5-monophosphate, as well as in pyrimidine nucleotide formation. Hence ...
) as a cosubstrate. This reaction is driven by the
hydrolysis Hydrolysis (; ) is any chemical reaction in which a molecule of water breaks one or more chemical bonds. The term is used broadly for substitution reaction, substitution, elimination reaction, elimination, and solvation reactions in which water ...
of
pyrophosphate In chemistry, pyrophosphates are phosphorus oxyanions that contain two phosphorus atoms in a linkage. A number of pyrophosphate salts exist, such as disodium pyrophosphate () and tetrasodium pyrophosphate (), among others. Often pyrophosphates a ...
. Orotidylate decarboxylase is located at the C-terminal domain of UMPS and converts this orotidylate intermediate into
uridine monophosphate Uridine monophosphate (UMP), also known as 5′-uridylic acid ( conjugate base uridylate), 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, th ...
(also referred to as uridylate or UMP) via
decarboxylation Decarboxylation is a chemical reaction that removes a carboxyl group and releases carbon dioxide (CO2). Usually, decarboxylation refers to a reaction of carboxylic acids, removing a carbon atom from a carbon chain. The reverse process, which is ...
. These two-steps are rapid and irreversible in mammals. In other
pyrimidine Pyrimidine (; ) is an aromatic, heterocyclic, organic compound similar to pyridine (). One of the three diazines (six-membered heterocyclics with two nitrogen atoms in the ring), it has nitrogen atoms at positions 1 and 3 in the ring. The oth ...
auxotrophs that do not have this bifunctional enzyme, usually less complex organisms, two separate enzymes are required to carry out this reaction. Both orotidylate and uridylate are major
pyrimidine Pyrimidine (; ) is an aromatic, heterocyclic, organic compound similar to pyridine (). One of the three diazines (six-membered heterocyclics with two nitrogen atoms in the ring), it has nitrogen atoms at positions 1 and 3 in the ring. The oth ...
nucleotide Nucleotides are Organic compound, 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 o ...
s, as uridylate is a precursor to RNA. Uridylate (UMP) is later converted to UDP via
phosphorylation In biochemistry, phosphorylation is described as the "transfer of a phosphate group" from a donor to an acceptor. A common phosphorylating agent (phosphate donor) is ATP and a common family of acceptor are alcohols: : This equation can be writ ...
by UMP kinase and ATP and then nucleoside diphosphate kinase reversibly
phosphorylates In biochemistry, phosphorylation is described as the "transfer of a phosphate group" from a donor to an acceptor. A common phosphorylating agent (phosphate donor) is ATP and a common family of acceptor are alcohols: : This equation can be writt ...
UDP to UTP. UTP can then be aminated through
catalysis Catalysis () is the increase in rate of a chemical reaction due to an added substance known as a catalyst (). Catalysts are not consumed by the reaction and remain unchanged after it. If the reaction is rapid and the catalyst recycles quick ...
by cytidine triphosphate synthetase to from CTP.


Enzyme mechanism

The reaction of
orotic acid 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. The compound is synthesized in the body via ...
(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 In thermodynamics, the Gibbs free energy (or Gibbs energy as the recommended name; symbol is a thermodynamic potential that can be used to calculate the maximum amount of work, other than pressure–volume work, that may be performed by a ther ...
of -8.3 + 0.5 kcal/mol. Two main interactions attract PRPP to assist orotate phophoribosyltransferase in this reaction. First, orotate phophoribosyltransferase has
Aspartic acid Aspartic acid (symbol Asp or D; the ionic form is known as aspartate), is an α-amino acid that is used in the biosynthesis of proteins. The L-isomer of aspartic acid is one of the 22 proteinogenic amino acids, i.e., the building blocks of protei ...
-
Aspartic acid Aspartic acid (symbol Asp or D; the ionic form is known as aspartate), is an α-amino acid that is used in the biosynthesis of proteins. The L-isomer of aspartic acid is one of the 22 proteinogenic amino acids, i.e., the building blocks of protei ...
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. The stabilization occurs through a hydrogen bonding network of these
hydroxyl In chemistry, a hydroxy or hydroxyl group is a functional group with the chemical formula and composed of one oxygen atom covalently bonded to one hydrogen atom. In organic chemistry, alcohols and carboxylic acids contain one or more hydroxy ...
groups with
pyrophosphate In chemistry, pyrophosphates are phosphorus oxyanions that contain two phosphorus atoms in a linkage. A number of pyrophosphate salts exist, such as disodium pyrophosphate () and tetrasodium pyrophosphate (), among others. Often pyrophosphates a ...
, water and magnesium. Second, the side-chains of the C-terminal end of the PRPP-binding motif interact favorably with PRPP’s 5-phosphate. In B. subtilis, PRPP is bound to these two sites with a Kd of 33mM. When
orotate 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. The compound is synthesized in the body via ...
is present,
pyrophosphate In chemistry, pyrophosphates are phosphorus oxyanions that contain two phosphorus atoms in a linkage. A number of pyrophosphate salts exist, such as disodium pyrophosphate () and tetrasodium pyrophosphate (), among others. Often pyrophosphates a ...
binding affinity is increased fourfold and the reaction undergoes
burst kinetics Burst may refer to: *Burst mode (disambiguation), a mode of operation where events occur in rapid succession **Burst transmission, a term in telecommunications **Burst switching, a feature of some packet-switched networks **Bursting, a signaling mo ...
, with rapid phosphoribosyl transfer and then slow release of products. 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. The loop opening happens in two-steps with the PRPP dissociation unfavorable and slow since the loop closes 85% of the time. Three key
pyrimidine Pyrimidine (; ) is an aromatic, heterocyclic, organic compound similar to pyridine (). One of the three diazines (six-membered heterocyclics with two nitrogen atoms in the ring), it has nitrogen atoms at positions 1 and 3 in the ring. The oth ...
nucleoside Nucleosides are glycosylamines that can be thought of as nucleotides without a phosphate group. A nucleoside consists simply of a nucleobase (also termed a nitrogenous base) and a five-carbon sugar (ribose or 2'-deoxyribose) whereas a nucleotid ...
s include
uridine 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 nuc ...
,
cytidine Cytidine (symbol C or Cyd) is a nucleoside molecule that is formed when cytosine is attached to a ribose ring (also known as a ribofuranose) via a β-N1-glycosidic bond. Cytidine is a component of RNA. It is a white water-soluble solid that is ...
and
thymidine Thymidine (nucleoside#List of nucleosides and corresponding nucleobases, symbol dT or dThd), also known as deoxythymidine, deoxyribosylthymine, or thymine deoxyriboside, is a pyrimidine nucleoside, deoxynucleoside. Deoxythymidine is the DNA nuc ...
, and they play major roles in nucleic acid biosynthesis as well as carbohydrate and lipid metabolism. 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. Like other pyrimidine phosphoribosyltransferases, orotate phosphoribosyltransferase has a flexible loop that moves to position groups in the ideal positions for
catalysis Catalysis () is the increase in rate of a chemical reaction due to an added substance known as a catalyst (). Catalysts are not consumed by the reaction and remain unchanged after it. If the reaction is rapid and the catalyst recycles quick ...
. They also use many water molecules to hold everything in place during the reaction.


Enzyme structure

The crystal structure of OPRTase has been solved several times by various scientific groups. 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.; Orotate phosphoribosyltransferase has a core part plus a flexible loop, which when closed prevents solvent from entering during reaction. In other organisms such as mammals, insects and slime modes it is one of the domains of
UMP synthase The enzyme Uridine monophosphate synthase (, UMPS) (orotate phosphoribosyl transferase and orotidine-5'-decarboxylase) catalyses the formation of uridine monophosphate (UMP), an energy-carrying molecule in many important biosynthetic pathways. In ...
, with the other being orotidylate decarboxylase. 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.
Orotic acid 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. The compound is synthesized in the body via ...
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 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. The compound is synthesized in the body via ...
, as well as Thr 128, Ala 129, Gly 130, Ala 132, Asp 124, Lys 26 and Lys 73 to PRPP. When Lys 26 is mutated, orotate phosphoribosyltransferase often exhibits reduced activity and specificity.


Disease relevance

Defects in orotate phosphoribosyltransferase have been implicated in numerous medical conditions. Defects in the orotate phosphoribosyltransferase domain of UMPS cause
orotic aciduria Orotic aciduria (AKA hereditary orotic aciduria) is a disease caused by an enzyme deficiency, resulting in a decreased ability to synthesize pyrimidines. It was the first described enzyme deficiency of the ''de novo'' pyrimidine synthesis pathway. ...
in humans, which is a rare hereditary metabolic disease resulting from problems with
pyrimidine Pyrimidine (; ) is an aromatic, heterocyclic, organic compound similar to pyridine (). One of the three diazines (six-membered heterocyclics with two nitrogen atoms in the ring), it has nitrogen atoms at positions 1 and 3 in the ring. The oth ...
metabolism. It can lead to
megaloblastic anemia Megaloblastic anemia is a type of macrocytic anemia. An anemia is a red blood cell defect that can lead to an undersupply of oxygen. Megaloblastic anemia results from inhibition of DNA replication, DNA synthesis during red blood cell production. ...
and orotic acid crystalluria, which is associated with physical and mental impairments.
Orotic aciduria Orotic aciduria (AKA hereditary orotic aciduria) is a disease caused by an enzyme deficiency, resulting in a decreased ability to synthesize pyrimidines. It was the first described enzyme deficiency of the ''de novo'' pyrimidine synthesis pathway. ...
was first reported in 1959 when excess
orotic acid 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. The compound is synthesized in the body via ...
was found in the urine of an infant. When individuals have a mutation leading to loss of orotate phosphoribosyltransferase activity and thus UTP production,
orotic acid 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. The compound is synthesized in the body via ...
builds up and can be as high as 1.5g/day in infant urine. This is because UTP is the normal end product in healthy individuals in the pyrimidine synthetic pathway and normally regulates the pathway.
Orotic acid 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. The compound is synthesized in the body via ...
buildup can lead to precipitation in the kidney and eventually renal failure. Similarly, in Holstein cattle, UMPS deficiency is caused by an autosomal disorder which leads to death of offspring in the early
embryo An embryo ( ) is the initial stage of development for a multicellular organism. In organisms that reproduce sexually, embryonic development is the part of the life cycle that begins just after fertilization of the female egg cell by the male sp ...
nic state. 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. For instance, one study found that the ratio of gene expression of orotate phosphoribosyltransferase to
dihydropyrimidine dehydrogenase In enzymology, a dihydropyrimidine dehydrogenase (NADP+) () is an enzyme that catalysis, catalyzes the chemical reaction :5,6-dihydrouracil + NADP+ \rightleftharpoons uracil + NADPH + H+ Thus, the two substrate (biochemistry), substrates of th ...
affects the prognosis of metastatic colorectal cancer patients after fluropyrimidine-based
chemotherapy Chemotherapy (often abbreviated chemo, sometimes CTX and CTx) is the type of cancer treatment that uses one or more anti-cancer drugs (list of chemotherapeutic agents, chemotherapeutic agents or alkylating agents) in a standard chemotherapy re ...
. 5-F-UMP is thought to become a suicide inhibitor for thymidylate synthetase and plays an important role in tumor growth inhibition. When resectable colorectal cancer patients were treated with oral 5-flurouracil, patients with high levels of orotate phosphoribosyltransferase had significantly better survival outcomes. Similarly prognosis potential based on orotate phosphoribosyltransferase levels and activity have been implicated in
bladder carcinoma Bladder cancer is the abnormal growth of cells in the bladder. These cells can grow to form a tumor, which eventually spreads, damaging the bladder and other organs. Most people with bladder cancer are diagnosed after noticing blood in their ...
and
gastric carcinoma Stomach cancer, also known as gastric cancer, is a malignant tumor of the stomach. It is a cancer that develops in the lining of the stomach. Most cases of stomach cancers are gastric carcinomas, which can be divided into a number of subtypes ...
.


See also

* Orotidylate decarboxylase *
UMP synthase The enzyme Uridine monophosphate synthase (, UMPS) (orotate phosphoribosyl transferase and orotidine-5'-decarboxylase) catalyses the formation of uridine monophosphate (UMP), an energy-carrying molecule in many important biosynthetic pathways. In ...
*
Pyrimidine Pyrimidine (; ) is an aromatic, heterocyclic, organic compound similar to pyridine (). One of the three diazines (six-membered heterocyclics with two nitrogen atoms in the ring), it has nitrogen atoms at positions 1 and 3 in the ring. The oth ...
Biosynthesis Biosynthesis, i.e., chemical synthesis occurring in biological contexts, is a term most often referring to multi-step, enzyme-Catalysis, catalyzed processes where chemical substances absorbed as nutrients (or previously converted through biosynthe ...
*
Orotic acid 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. The compound is synthesized in the body via ...
* PRPP


References

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