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Misunderstanding about nucleotide biosynthesis

Misunderstanding about nucleotide biosynthesis


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Some months ago, I asked what was a phosphoester bond, because I didn't really understand the following picture and explanations provided in the "Molecular biology of the gene" from Watson, Baker, Bell and al. (7th edition).

The explanation is the following:

We can think of how the base is joined to 2'-deoxyribose by imagining the removal of a molecule of water between the hydroxyl on the 1' carbon of the sugar and the base to form a glycosidic bond (Fig. 4-2). The sugar and base alone are called a nucleoside. Likewise, we can imagine linking the phosphate to 2'-deoxyribose by removing a water molecule from between the phosphate and the hydroxyl on the 5' carbon to make a 5' phosphomonoester. Adding a phosphate (or more than one phosphate) to a nucleoside creates a nucleotide.

Thus, by making a glycosidic bond between the base and the sugar, and by making a phosphoester bond between the sugar and the phosphoric acid, we have created a nucleotide.

What I didn't understand months ago was, to resume, "why is the bond between the phosphate group and the 2'-deoxyribose called a phosphoester bond since the phosphoester bond (which is, by definition, a P-O bond) already exists before the bonding between the phosphate group and the sugar?"

While this question is still bothering me (I was not entirely satisfied with the answers I got back then, because the synthesis of a single strand of DNA is something which is probably different from the synthesis of nucleotides themselves), the question I'm asking myself right now is: "why do general biology books always talk about condensation reactions, while this article and this book do not mention it in the biosynthesis pathways of nucleotides?"

What I mean is: it seems like the biosynthesis of nucleotides is far more complicated than just being two condensation reactions. Furthermore, when I look at the 10th step of this picture (which comes from the article I mentioned above), I see a condensation reaction at the end of the pathway, but it does not occur where the book from Watson and al. suggests it should:

So, after months of wondering and researchs about all this, my final question about this is, as mentioned above:

Why do biology books always mention condensation reactions while, apparently, it doesn't have a lot to do with the manner in which the nucleotides are synthesized?


A condensation reaction is defined on Wikipedia as

a reaction in which two molecules or moieties, often functional groups, combine to form a larger molecule, together with the loss of a small molecule. Possible small molecules that are lost include water, acetic acid, hydrogen chloride, or methanol, but most commonly in biological reactions it is water.

It doesn't say anything about the number of steps required to reach the final compound, nor about the type of bonds formed. Following this definition, the nucleotide synthesis occurs via condensation reactions.


CATABOLISM OF PURINE NUCLEOTIDES

J. Frank Henderson , A.R.P. Paterson , in Nucleotide Metabolism , 1973

I Introduction

Purine nucleotides are broken down by animal cells to fragments which are excreted in order to maintain a relatively constant internal composition in the face of a constant synthesis of these compounds both de novo and from dietary constituents. Microorganisms may also catabolize nucleotides, in some cases as sources of energy, carbon, and nitrogen for growth. Although multiple pathways for nucleotide catabolism exist in animals, only certain of these exist in any particular cell. In addition, when alternative pathways do exist within the same cell, it is sometimes very difficult to evaluate the relative importance of each. The overall picture of potential pathways of purine nucleotide catabolism as given in summary in this chapter, therefore, cannot be applied indiscriminately to all cells.


Nucleotide Structure

Nucleotide structure is simple, but the structure they can form together is complex. Below is an image of DNA. This molecule consists of two strands which wrap around each other, forming hydrogen bonds in the middle of the structure for support. Each nucleotide within has a specific structure which enables this formation.

Nitrogenous base

The nitrogenous base is the central information carrying part of the nucleotide structure. These molecules, which have different exposed functional groups, have differing abilities to interact with each other. As in the image, the idea arrangement is the maximum amount of hydrogen bonds between nucleotides involved. Because of the structure of the nucleotide, only a certain nucleotide can interact with other. The image above shows thymine bonding to adenine, and guanine bonding to cytosine. This is the proper and typical arrangement.

This even formation causes a twist in the structure, and is smooth if there are no errors. One of the ways proteins are able to repair damaged DNA is that they can bind to uneven spots within the structure. Uneven spots are created when hydrogen bonding does not occur between the opposing nucleotide molecules. The protein will cut out one nucleotide, and replace it with another. The duplicate nature of the genetic strands ensures that errors like this can be corrected with a high degree of accuracy.

Sugar

The sugar, with its exposed oxygen, can bond with the phosphate group of the next molecule. They then form a bond, which becomes the sugar-phosphate backbone. This structure adds rigidity to the structure, as the covalent bonds they form are much stronger than the hydrogen bonds between the two strands. When proteins come to process and transpose the DNA, they do so by separating the strands and reading only one side. When they pass on, the strands of genetic material comes back together, driven by the attraction between the opposing nucleotide bases. The sugar-phosphate backbone stays connected the whole time.

Phosphate Group

The last part of nucleotide structure, the phosphate group, is probably familiar from another important molecule ATP. Adenosine triphosphate, or ATP, is the energy molecule that most life on Earth relies upon to store and transfer energy between reactions. ATP contains three phosphate groups, which can store a lot of energy in their bonds. Unlike ATP, the bonds formed within a nucleotide are known as phosphodiester bonds, because they happen between the phosphate group and the sugar molecule.

During DNA replication, an enzyme known as DNA polymerase assembles the correct nucleotide bases, and begins organizing them against the chain it is reading. Another protein, DNA ligase, finished the job by creating the phosphodiester bond between the sugar molecule of one base and the phosphate group of the next. This creates the backbone of a new genetic molecule, able to be passed to the next generation. DNA and RNA contain all the genetic information necessary for cells to function.


Nucleotides Metabolism and De Novo Synthesis of Nucleotides

Nucleotide consists of a purine or pyrimidine base plus a pentose sugar (ribose or deoxyribose) and a phosphoryl group (H3PO4). The purine ring consists of a 5-membered imidazol ring fused to a six-membered ring structure with two common or bridge carbon atoms (C-4 and C-5) and contains 4-N atoms. The pyrimidine ring has a simpler struc­ture with only a six-membered ring with two N-atoms.

The metabolism of nucleotides includes synthesis, inter-conversions, and catabolism of various purine and pyrimidine nucleotides which are schematically shown in Fig. 9.32 and 9.33 respectively. Metabolism of nucleotides is clearly known in animals than in plant cells.

Purine and pyrimidine nucleotides can be synthesized in living organisms either by (i) de novo pathways, or (ii) salvage pathways. In de novo pathways, the synthesis of nucleotides begins with their metabolic precursors: amino acids, ribose-5-phosphate, CO2, and NH3. In salvage pathways, the breakdown products of nucleotides i.e. free bases and nucleosides are salvaged and recycled back to synthesize nucleotides again.

Salvage pathways may involve reconstruction of nucleotides from free bases by addition of ribose-phosphate moiety, or by phos­phorylation of nucleosides. De novo pathways are more important quantitatively than salvage path­ways. However, by using salvage pathways for nucleotides synthesis, the cells do conserve energy.

De Novo Synthesis of Purine Nucleotides (IMP, AMP & GMP):

i. Free bases are not intermediates in de novo pathways of nucleotides synthesis i.e., they are not synthesized and then attached to ribose phosphate.

ii. The purine ring structure is built up one or a few atoms at a time, and is gradually attached to ribose phosphate throughout the process.

iii. Phosphoribosyl pyrophosphate (PRPP), is an important intermediate and the starting point in purine nucleotide synthesis. It is formed from α-D-Ribose-5-phosphate (Fig. 9.34)

iv. (The ultimate precursors of the purine ring are shown in Fig. 9.35. These precursors were established from information obtained from isotopic experiments with 14 C or 15 N- labeled precursors that were administered into pigeons and tracing the incorporation of labeled atoms into the purine ring of their excreted uric acid).

iv. IMP (Inosine monophosphate or Inosinate) is the first purine nucleotide to be synthesized.

v. IMP is then converted into AMP and GMP.

Formation of IMP from PRPP:

a. Synthesis of IMP from PRPP (phosphoribosyl pyrophosphate) takes place in ten differ­ent steps as shown in Fig. 9.36.

b. The 5-membered imidazol ring is added first to PRPP the remaining six-membered ring of purine is built up afterwards.

(i) In the first committed step of this pathway, phosphoribosylamine is formed by the action of the enzyme glutamine phosphoribosyl pyrophosphate amidotransferase. An amino group supplied by glutamine is attached to C-1 of PRPP and there is inversion of configuration at C-1, from a to p position. The purine ring is subsequently built on this structure. Atom no. 9 (N-9) of the purine ring is introduced in this first step.

(ii) In the second step, the enzyme synthetase forms an amide bond between carboxyl group of glycine and amino group of phosphoribosylamine forming 5′-phosphoribosyl glycinamide. ATP is hydrolyzed to provide energy. The atoms 4, 5 and 7 of the purine ring are introduced in this step.

(iii) In the third step, C-8 of the purine ring is introduced as a formyl group donated by 10-formyl tetrahydrofolate, in presence of the enzyme formyl transferase, so that 5′- phosphoribosyl-N-formyl glycinamide is formed.

(iv) In the fourth step, N-3 of purine ring is introduced by transfer of another amino group from glutamine to phosphoribosyl formyl glycinamide by a synthetase enzyme, form­ing 5′-phosphoribosyl-N-formyl glycinamidine. ATP is hydrolyzed and provides en­ergy.

(v) In the fifth step, cyclization reaction occurs in presence of synthetase, Mg 2+ , and K + ions, so that imidazol ring is closed. The product is 5′-phosphoribosyl-5- aminoimidazole.

(vi) In the sixth step, C-6 of the purine ring is introduced by addition of bicarbonate (CO2 + H2O → HCO3 – ) in presence of a specific carboxylase enzyme. The product of this reaction is 5′-phosphoribosyl-5-aminoimidazole-4-carboxylate.

(vii) In the seventh step, N-8 of the purine ring is contributed by aspartate. The latter forms an amide with 4-carboxyl group in presence of synthetase, and a succinocarboxamide is formed. ATP is hydrolyzed and provides energy.

(viii) 5′-phosphoribosyl – 4 – (N-succino carboxamide) – 5-aminoimidazole is now cleaved in presence of adenylosuccinate lyase to release formate and forming 5′-phosphoribosyl- 4-carboxamide – 5 – aminoimidazole.

(ix) In the ninth step, the final atom of purine ring (i.e., C-2) is introduced which is supplied by a formyl group from 10-formyl tetrahydrofolate to the 5-amino group of the almost completed ribonucleotide.

(x) In the last step, a second ring closure takes place by elimination of water to form IMP. The enzyme involved is IMP-cyclohydrolase (IMP-synthase)

Synthesis of IMP from ribose-5-phosphate requires a total of six high energy phos­phate groups from ATPs (assuming hydrolysis of pyrophosphate (P-P)) released in step (i).

Conversion of IMP into AMP and GMP:

a. IMP is converted into AMP (adenosine mono-phosphate) and GMP (guanosine monophosphate) by two different pathways, each consisting of two steps.

(i) Conversion of IMP into AMP:

(a) In the first step of this pathway, keto group of IMP is first displaced by the amino of aspartate to produce adenylosuccinate in the presence of the enzyme synthetase. GTP is hydrolyzed and provides energy.

(b) Adenylosuccinate is now cleaved non-hydrolytically by the enzyme adenylosuccinate lyase to produce fumarate and the purine nucleotide AMP (adenosine mono-phosphate.)

(ii) Conversion of IMP into GMP:

(a) In the first step of this pathway, there is dehydrogenation of IMP to xanthosine- 5-phosphate (XMP), in the presence of NAD + -dependent IMP-dehydrogenase.

(b) The second step involves transfer of an amino group from glutamine to C-2 of the xanthine ring to produce GMP (guanosine monophosphate). ATP is hydrolyzed to provide energy, while glutamate is released.

b. (After the formation of purine mononucleotides, purine di and tri-nucleotides may be synthe­sized by addition of one or two more phosphoryl groups respectively).

De Novo synthesis of Pyrimidine Nucleotides:

I. Common pyrimidine ribonucleotides are cytidine-5′-monophosphate (CMP or cytidylate) and uridine-5′-monophosphate (UMP or uridylate).

II. De novo biosynthesis of pyrimidine nucleotides is simpler than those of purine nucleotides because of the simpler structure of pyrimidine ring.

III. In contrast to the de novo biosynthetic pathway of purine nucleotides, in pyrimidine biosynthetic pathway the pyrimidine ring is constructed before ribose – 5 – phosphate is incorporated into the nucleotide.

IV. Orotidine-5′-monophosphate (OMP), is the first pyrimidine nucleotide to be synthesized. From OMP, pathways lead to synthesis of nucleotides of uracil, cytosine and thymine.

De novo synthesis of pyrimidine nucleotides is illustrated in Fig. 9.37, a brief description of this follows:

(i) The first step in this pathway is the synthesis of carbamoyl phosphate from CO2 and NH4 + by carbamoyl phosphate from CO2 and NH4 + by carbamoyl phosphate synthetase. NH4 + is supplied by glutamine.

(ii) In the next step, carbamoyl phosphate reacts with aspartate to form carbamoyl aspartate. The reaction is catalyzed by aspartate carbamoyl transferase.

(iii) In the third step, the pyrimidine ring is closed by dehydroorotase to form dihydroorotate with elimination of water molecule.

(iv) Dihydroorotate is now oxidized to orotate by dehydrogenase enzyme.

(v) In the fifth step, orotate is converted into orotidine-5′-monophosphate (OMP) by the enzyme orotate phosphoribosyl transferase. The ribose phosphate moiety is supplied by PRPP (phosphoribosyl pyrophosphate).

(vi) In the last reaction, OMP is decarboxylated by OMP-decarboxylase to yield UMP (uridine monophosphate).

(v) UMP is the precursor of other pyrimidine nucleotides. First, UMP is phosphorylated to UTP (uridine triphosphate). Then, CTP (cytidine triphosphate) is formed from UTP by the action of the enzyme cytidylate synthetase. ATP is hydrolyzed and provides energy NH4 + is supplied by glutamine (Gln) which is converted into glutamate (Glu). See Fig. 9.37.

Formation of Deoxy Ribonucleotides:

a. Deoxy nucleotides are formed by reduction of corresponding ribonucleotides by ribonucleotide reductases, 2′-OH group of the ribopentose sugar is replaced with hy­drogen.

b. Thymidylate (TMP) is formed from dUMP

TMP is synthesized in the cells from dUMP (deoxy uridine monophosphate) and the latter can be formed by two different pathways:

(i) Mainly, by deamination of dCMP (deoxycytidine-monophosphate) in the presence of the enzyme deoxy-cytidylate deaminase.

(ii) Also, by reduction of UDP to dUDP followed by phosphorylation of dUDP to dUTP. The latter is then hydrolyzed to dUMP.

c. dUMP is now methylated to from thymidylate (TMP) by the enzyme thymidylate synthase. The methyl group is provided by 5, 10-methylene tetrahydrofolate.

Catabolism (Degradation) Of Nucleotides:

(1) Catabolism of Purine Nucleotides:

The Pathway of degradation of purine nucleotides is illustrated in Fig. 9.38. In primates, birds, and some other animals, the end product of this pathway is uric acid.

a. Uric acid is further catabolized to other excretory products in different groups of other animals (Fig. 9.39). Most mammals other than primates oxidize uric acid further to allantoin. In many other animals, allantoin is catabolized further to allantoic acid (as in bony fishes), urea (as in amphibians and cartilaginous fishes), or ammonia and CO2 (as in marine invertebrates).

b. No ATPs are formed during catabolism of purine nucleotides.

(2) Catabolism of Pyrimidine Nucleotides:

I. Ribose phosphate is released during catabolism prior to destruction of base.

II. Pyrimidines are catabolized to β-alanine, NH3, and CO2.

III. Thymine is catabolized to β-aminoisobutyrate.

IV. As in case of catabolism of purine nucleotides, no ATPs are formed in pyrimidine nucleotides catabolism.


Human Milk

Dolly Sharma , . Pearay Ogra , in Mucosal Immunology (Fourth Edition) , 2015

Nucleotides

Nucleotides are present in human milk, making up about 2–5% of its total nonprotein nitrogen, which is greater than in ruminants ( Cosgrove, 1998 ). The nucleotides amount to 53–58 mg/L in human colostrum and about 33 mg/L in mature milk ( Kuchan et al., 1998 ). Nucleotides participate in several biochemical processes and may support the breast-fed offspring in various ways. They function as building blocks of nucleic acids. This may be especially important for the very rapid early growth of the infant’s immune system, which expands primarily in response to exposure to colonizing microbes, particularly on the mucosa in the gut.

Nucleotides are also important in various biosynthetic pathways, by transferring chemical energy, as coenzyme components and as biologic regulators. The maturation of the gut mucosa may be enhanced by nucleotides. Enzymes capable of limited digestion of nucleotides are present in human milk, and fetal intestine homogenate could, when incubated with human milk, add to such degradation ( Thorell et al., 1996 ). Some studies have shown the advantage of adding nucleotides to formula to try to achieve some of the effects of nucleotides in human milk. Thus, such addition to formula in premature infants resulted in higher serum levels of IgA and IgM ( Navarro et al., 1999 ) greater responses to immunization against H. influenzae type b, tetanus, and diphtheria toxoid ( Schaller et al., 2004 ) higher numbers of natural killer (NK) cells and activity ( Carver et al., 1991 ) and less ( Pickering et al., 1998 Ostrom et al., 2002 ) diarrheal disease ( Gutierrez-Castrellon et al., 2007 ) ( Brunser et al., 1994 ). Long-term breast-feeding has resulted in higher serum antibody responses to oral poliovirus vaccine compared with a formula plus nucleotide or a formula group ( Pickering et al., 1998 ). Compared with healthy term infant subjects fed a control formula, those fed formula fortified with nucleotides had a reduced risk of developing diarrhea and had significantly higher serum concentrations of IgA over the next 48 weeks ( Yau et al., 2003 ).


FOLATE AND METHIONINE CYCLES

The &ldquocore&rdquo part of the one-carbon metabolism comprises the Folate and Methionine cycles, which are linked together. These two cycles integrate cell nutrient status using 1C-groups from glycine and serine as &ldquoinputs&rdquo to generate different &ldquooutputs&rdquo such as nucleotides, glutathione, SAM, and other metabolites, which are required for DNA and RNA biosynthesis, as well as for the maintenance of the redox and epigenetic cell states.

Folate cycle

Folates are referred to the family of B9 vitamins [33]. They are naturally present in different sources of food or can be synthesized chemically (e.g. folic acid) as dietary supplements. Folates function as carriers that distribute one-carbon groups from &ldquoinputs&rdquo to &ldquooutputs&rdquo (Figure ​(Figure11 and ​and2).2). Once transported to the cell, the vitamin undergoes covalent modification by polyglutamination. It is further substituted by the one-carbon moiety in the N5 and/or N10 position at different oxidation levels: formate (10-formylTHF), formaldehyde (5,10-methyleneTHF), or methanol (5-methylTHF) [34].

Folate cycle, its &ldquooutputs&rdquo and the energy balance

Critical enzymes of the Folate cycle are shown. TetraHydroFolate (THF) is a carrier that distributes one-carbon groups (1C-group) from serine to different &ldquooutputs&rdquo &ndash thymidylates, purines, SAM, GSH, etc (shown in black boxes). After accepting the 1C-group, THF undergoes modifications that alter its oxidation states: 10-formylTHF, 5,10-methyleneTHF, 5-methylTHF (shown in different background colors). Donated carbon and nitrogen atoms corresponding to their numbers in the pyrimidine and purine rings are shown in brackets. Red asterisks indicate the enzymes that are currently being explored as drug targets. Enzymes marked with orange asterisks are considered as potential drug targets. Folate cycle can provide cells with additional source of energy. Two molecules of NADPH are synthesized in cytoplasm in reactions catalyzed by DHFR (conversion of DHF to THF) and MTHFD1 (conversion of 5,10-methylenTHF to 5,10-methenylTHF), as well as in mitochondria by MTHFD2L (conversion of 5,10-methylenTHF to 5,10-methenyl THF). One molecules of NADPH is used by MTHFR which links Folate cycle to the Methionine cycle. Also, ATP can be synthesized during MTHFD1- (cytoplasm) or MTHFD1L-mediated (mitochondria) conversion of 10-formylTHF to THF.

As mentioned above, there are only two direct sources of 1C-groups in one-carbon metabolism &ndash serine and glycine. Thus, the central reaction of the Folate cycle is conversion of serine to glycine by SHMT1 and SHMT2 enzymes. By transferring the 1C-group from serine and THF, this reaction generates 5,10-methyleneTHF &ndash the first donor of one-carbon group in the folate cycle. Another source of 5,10-methyleneTHF comes from the enzymatic cleavage of glycine by an enzyme called glycine decarboxylase (GLDC), which resides in mitochondria.

In turn, 5,10-methyleneTHF can be used in three ways (Figure ​(Figure2).2). First, it can serve as 1C-donor for the initial step of thymidylate biosynthesis, a reaction catalyzed by thymidylate synthase (TS). In this reaction 5,10-methyleneTHF provides one-carbon group for the pyrimidine biosynthesis and is oxidized into dihydrofolate (DHF). In the next reaction dihydrofolate reductase (DHFR) reduces DHF to THF enclosing this metabolic loop.

Second, 5,10-methyleneTHF can be used by a cytosolic enzyme Methylenetetrahydrofolate reductase 1 (MTHFD1), or mitochondrial tandem enzymes Methylenetetrahydrofolate reductases MTHFD2L/MTHFD2, to generate 10-formylTHF. 10-formylTHF is a 1C-donor for the two reactions of purine biosynthesis catalyzed by Trifunctional enzyme Phosphoribosylglycinamide Formyltransferase/ Synthetase/ Phosphoribosylaminoimidazole Synthetase (GART) and Bifunctional 5-Aminoimidazole-4-Carboxamide Ribonucleotide Formyltransferase/IMP Cyclohydrolase (ATIC), both of which in turn generate THF.

Third, 5,10-methyleneTHF is used by Methylentetrahydrofolatereductase (MTHFR) to generate methylTHF. The latter donates a methyl group to homocycteine resulting in the formation of methionine and THF. By this way the Folate cycle is coupled with Methionine cycle. Finally, THF is converted into 5,10-methyleneTHF by SHMT1 and SHMT2 thus enclosing the Folate cycle.

Methionine cycle

Another arm of the 1C-metabolic process is the methionine cycle (Figure ​(Figure3).3). It starts with methionine synthesis from homocysteine and methylTHF catalyzed by methionine synthase (MS). Subsequently, methionine adenyltransferase (MAT) synthesizes SAM, the main donor of methyl groups in the cell. After demethylation, SAM is converted to S-adenosylhomocysteine (SAH). Finally, S-adenosyl homocysteine hydrolase (SAHH) mediates de-adenylation of SAHH resulting in homocysteine and full turn of the cycle.

Folate cycle is coupled with Methionine cycle

During Folate cycle MTHFR reduces 5,10-methyleneTHF to 5-methylTHF. Subsequently, 5-methylTHF donates its carbon group to convert homo-cysteine (hcystein) to methionine by methionine synthase (MS), hence initiating Methionine cycle. In turn, methionine is used by methionine adenosyltransferase (MAT) to generate S-adenosylmethionine (SAM) &ndash the principal donor of methyl groups for DNA and proteins methylation. Thus, SAM is used by different methyltransferases, resulting in S-adenosylhomocysteine after its demethylation. Finally, S-adenosylhomocysteine hydrolase (SAHH) mediates deadenylation of S-adenosylhomocysteine to hcysteine, enclosing the methionine cycle. Homocysteine can be used by cystathionine synthase (CBS), which converts it to cystathionine. In turn, cystathionine is a substrate for cystathionine gamma-lyase (CTH), which uses it for synthesis of cysteine. Cysteine is required for the synthesis of proteins as well as for generation of taurine and glutathione, the latter is one of the critical molecules for redox homeostasis.


Molecular biology of pyridine nucleotide biosynthesis in Escherichia coli. Cloning and characterization of quinolinate synthesis genes nadA and nadB

The two genes, nadA and nadB, responsible for quinolinate biosynthesis from aspartate and dihydroxyacetone phosphate in Escherichia coli were cloned and characterized. Quinolinate (pyridine-2,3-dicarboxylate) is the biosynthetic precursor of the pyridine ring of NAD. Gene nadA was identified by complementation in three different nadA mutant strains. Sequence analysis provided an 840-bp open reading frame coding for a 31,555-Da protein. Gene nadB was identified by complementation in a nadB mutant strain and by the L-aspartate oxidase activity of its gene product. Sequence analysis showed a 1620-bp open reading frame coding for a 60,306-Da protein. For both genes, promoter regions and ribosomal binding sites were assigned by comparison to consensus sequences. The nadB gene product, L-aspartate oxidase, was purified to homogeneity and the N-terminal sequence of 19 amino acids was determined. The enzyme was shown to be specific for L-aspartate. High-copy-number vectors, carrying either gene nadA, nadB or nadA + nadB, increased quinolinate production 1.5-fold, 2.0-fold and 15-fold respectively. Both gene products seem to be equally rate-limiting in quinolinate synthesis.


Purine salvage reactions

Not all nucleotides in a cell are made from scratch. The alternatives to de novo syntheses are salvage pathways. Salvage reactions to make purine nucleotides start with attachment of ribose to purine bases using phosphoribosylpyrophosphate (PRPP).

The enzyme catalyzing this reaction is known as hypoxanthine/guanine phosphoribosyltransferase (HGPRT - Figure 6.175) and is interesting from an enzymological as well as a medical perspective. First, the enzyme is able to catalyze both of the next two important salvage reactions - converting hypoxanthine to IMP or guanine to GMP.

HGPRT is able to bind a variety of substrates at its active site and even appears to bind non-natural substrates, such as acyclovir preferentially over its natural ones.

From a medical perspective, reduction in levels of HGPRT leads to hyperuricemia, a condition where uric acid concentration increases in the body. Complete lack of HGPRT is linked to Lesch-Nyhan syndrome, a rare, inherited disease in high uric acid concentration throughout the body is associated with severe accompanying neurological disorders.

Reduced production of HGPRT occurs frequently in males and has a smaller consequence (gout) than complete absence. Interestingly, gout has been linked to a decreased likelihood of contracting multiple sclerosis, suggesting uric acid may help prevent or ameliorate the disease.

Expression of HGPRT is stimulated by HIF-1, a transcription factor made in tissues when oxygen is limiting, suggesting a role for HGPRT under these conditions.

The enzyme known as adenine phosphoribosyltransferase (APRT) catalyzes the reaction corresponding to HGPRT for salvaging adenine bases.


Misunderstanding about nucleotide biosynthesis - Biology

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BRANCHED-CHAIN AMINO ACID CATABOLISM IN MUSCLE IS CONNECTED TO THE PURINE NUCLEOTIDE CYCLE

Branched-chain amino acids (BCAA) 1 1 The abbreviations used are: BCAA, branched-chain amino acids BCKA, branched-chain ketoacid. play an important role in the generation of glutamine because the net result of their metabolism in muscle is the transfer of amino nitrogen (in the form of aspartate) from muscle proteins into the purine nucleotide cycle. BCAAs (valine, isoleucine, and leucine) make up about 35% of the essential amino acids in muscle proteins and about 40% of the preformed amino acids required by mammals [ 16 ]. Because muscle is a major organ for the transamination of these amino acids, BCAAs are a major source of the amino nitrogen for both alanine and glutamine production in the muscle. Glutamate generated from transamination of BCAAs is subsequently used to generate aspartate by the transamination of oxalacetate, a reaction catalyzed by aspartate aminotransferase (Fig. 3). As previously discussed (Fig. 2), the oxalacetate is most likely formed from fumarate. When aspartate enters the purine nucleotide cycle, its amino nitrogen leaves the cycle as ammonia, which is used to synthesize glutamine that leaves the muscle.

Alanine, which is formed from the transamination of glutamate with pyruvate, also leaves the muscle and is transported to the liver where it is converted to glucose. This view is consistent with arterio-venous difference measurements [ 14 ] referred to previously and provides an integrated view of the relationship between catabolism of protein in muscle and the purine nucleotide cycle.

The transamination of BCAAs results in the formation of large quantities of branched-chain ketoacids (BCKAs) (α-ketoisocaproic acid, α-ketoisovaleric acid, and α-keto-β-methylvaleric acid). The BCKAs are then either oxidized in muscle [ 17 , 18 ] or are released from muscle and subsequently oxidized in the liver, beginning with the action of BCKA dehydrogenase complex. Indeed, endurance exercise promotes not only protein catabolism in muscle but also the oxidation of branched-chain α-keto acids [ 18 , 19 ]. The oxidation of the BCKAs of valine and isoleucine (i.e. α-ketoisovaleric acid and α-keto-β-methylvaleric acid) in muscle generates propionyl-CoA, which upon carboxylation and subsequent isomerization enters the tricarboxylic acid cycle as succinyl-CoA. This “feed in” can lead to the formation of fumarate and subsequently oxalacetate and in this manner can perform an anaplerotic function to the tricarboxylic acid cycle.


Misunderstanding about nucleotide biosynthesis - Biology

Introduction
Nucleotides play a variety of important roles in all cells. They are the activated precursors of DNA and RNA. ATP, an adenine nucleotide, is a universal currency of energy in biological systems. GTP is an essential carrier of chemical energy. Adenine nucleotides are components of the coenzymes NAD+, NADP+, FMN, FAD and Coenzyme A. UDP-Glucose in Glycogen synthesis and CDP-diacylglycerol in Phosphoglyceride synthesis are the nucleotide derivatives that act as activated intermediates. Cyclic AMP is a ubiquitous mediator for the action of many hormones. All cells can synthesize nucleotides from simple building blocks (de novo synthesis) or by the recycling of pre-formed bases (Salvage pathway). Nucleotides are phosphate esters of pentoses in which a nitrogenous base is linked to C1&rsquo of the sugar residue. A nucleotide without the phosphate group is known as a nucleoside. The major purine components of nucleic acids are adenine and guanine residues. The major pyrimidine residues are those of Cytosine, Uracil and Thymine. Pyrimidines are bound to ribose through N 1 atoms.

Synthesis of purine ribonucleotides
IMP is synthesized from ribose 5-phosphate. There are 11 reactions in the formation of IMP. IMP is converted to GMP and AMP with the help of ATP and GTP respectively. Nucleoside monophosphates are converted to nucleoside diphosphates by base specific monophosphate kinases. Purine nucleotide synthesis is regulated by feedback inhibitor &ndash AMP, GMP and IMP. An important regulatory factor is the availability of PRPP. Salvage pathway for purines is observed in RBC and the brain. Free purines are salvaged by APRTase and HGPRTase enzymes

Synthesis of pyrimidine ribonucleotides
Pyrimidine ring is synthesized as free pyrimidine and then it is incorporated into the nucleotide. 6 reactions are involved in the synthesis of UMP. UDP and UTP are synthesized from UMP with the help of ATP. CTP is formed by adding an amino group from glutamine. Pyrimidine can also be salvaged using PRPP. In orotic aciduria, excretion of large amount of orotic acid is observed. It results from the deficiency of either orotate phospho ribosyl transferase or OMP decarboxylase.

Formation of deoxyribonucleotides
Ribonucleotide reductase catalyzes the synthesis of deoxyribonucleotide. The reductant is NADPH. Thioredoxin transfers electrons from NADPH for reduction of 2&rsquo-OH of ribose. dTMP is formed by thymidylate synthase by methylation of deoxy uridine monophosphate.

Degradation of nucleotides
Nucleotides of a cell undergo continuous turnover. Purines are catabolized and the end product is uric acid. Gout is a disease characterized by elevated levels of uric acid in body fluids. Sodium urate crystals are precipitated in the joints and soft tissues to cause painful arthritis. In Lesch-Nyhan syndrome, HGPRT deficiency occurs, leading to excessive uric acid production through PRPP accumulation. Gout is treated by allopurinol administration. Animal cells degrade pyrimidine nucleotides to their component bases by dephosphorylation, deamination and glycosidic bond cleavages to give rise to carbon dioxide, ammonia, &beta-alanine and &beta-amino isobutyrate.

Nucleotide Coenzymes
Nucleotides are the components of many enzyme cofactors. Adenosine is a part of their structure in a variety of enzyme cofactors serving a wide range of chemical functions. Coenzyme A is synthesized from pantothenic acid and ATP.

Nucleotides play a variety of important roles in all cells. They are the activated precursors of DNA and RNA. ATP, an adenine nucleotide, is a universal currency of energy in biological systems. GTP is an essential carrier of chemical energy. Adenine nucleotides are components of the coenzymes NAD+, NADP+, FMN, FAD and Coenzyme A. IMP is synthesized from ribose 5-phosphate. There are 11 reactions in the formation of IMP. Nucleoside monophosphates are converted to nucleoside diphosphates by base specific monophosphate kinases. Purine nucleotide synthesis is regulated by feedback inhibitor &ndash AMP, GMP and IMP. Recycling of purines formed by the degradation of nucleotides is possible. Pyrimidine ring is synthesized as free pyrimidine and then it is incorporated into the nucleotide. Nucleotides of a cell undergo continuous turnover. Uric acid is the breakdown product of purine nucleotide. Gout is a disease characterized by elevated levels of uric acid in body fluids. Pyrimidines on degradation give rise to carbon dioxide, ammonia, &beta-alanine and &beta-amino isobutyrate.

  • Concept map depicting nucleotide synthesis from purines/pyrimidines.
  • Structures are shown with examples and animated.
  • Tabulation of nitrogenous bases, nucleosides and nucleotides.
  • Formation of IMP is explicitly shown with animated structures.
  • Flow chart of various compounds like UMP is described.
  • Degradation of purines and pyrimidines is discussed in detail.

Synthesis of Purine Ribonucleotides

  • Synthesis of inosine monophosphate
  • Synthesis of adenine and guanine ribonucleotides
  • Regulation of purine nucleotide biosynthesis
  • Salvage pathway for purines

Synthesis of Pyrimidine Ribonucleotides

  • Synthesis of uridine monophosphate
  • Synthesis of UTP and CTP
  • Regulation of pyrimidine nucleotide biosynthesis

Formation of Deoxyribonucleotides

Degradation of Nucleotides

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