Nucleotide Biosynthesis

Biosynthesis of Nucleotides

• Nucleotides serve as precursors to DNA and RNA.
• Nucleotides are carriers of chemical energy.
• Nucleotides are made via De novo and Salvage pathways
• De novo synthesis for purine and pyrimidine is identical in all organisms.
• Free bases are not intermediates in the de novo pathway, but are in the salvage process.
• PRPP serves as a key precursor for the de novo pathway.
• Amino acids are key components, with glutamine acting as a source of amino groups.

De Novo - Purines

• Adenosine 5'-monophosphate (AMP) and guanosine 5'-monophosphate (GMP) are parent nucleotides of nucleic acids.

Regulation of Purine Biosynthesis

• There are three major feedback mechanisms in place to regulating de novo purine nucleotide synthesis.

Pyrimidine Nucleotides

• Cytidine 5'-monophosphate (CMP) and uridine 5'-monophosphate (UMP) represent common pyrimidine ribonucleotides.
• CMP & UMP contain the pyrimidines cytosine and uracil.
• The first three enzymes are part of a single trifunctional protein in eukaryotes.

Pyrimidine Nucleotide Regulation

• Regulation of pyrimidine nucleotide synthesis is via aspartate transcarbamoylase (Atcase).
• The ATCase molecule has six catalytic and six regulatory subunits.

Nucleoside Monophosphate Conversion

• Nucleotides become nucleoside triphosphates
• AMP becomes ADP because of adenylate kinase
• ATP brings about other formations via ATP + NMP → ADP + NDP
• Nucleoside diphosphates are converted to triphosphates via NTPd + NDPa → NDPd + NTPa

Deoxyribonucleotide Synthesis

• Deoxyribonucleotides are the building blocks of DNA.
• A unique reaction turns ADP into 2’deoxyadenosine diphosphate (dADP) with ribonucleotide reductase.
• The process is a Reduction of D-ribose

The Role of Ribonucleotide Reductase

• Involves Deoxynucleoside Triphosphates

Ribonucleotide Reductase

• Serves as an example of free radical usage.

Purine and Pyrimidine Degradation

• Purine degradation produces uric acid.
• Pyrimidine degradation produces urea.

Purine and Pyrimidine Recycling

• Free purine and pyrimidine bases are recycled in the cell.
• Adenosine phosphoribosyltransferase is used
• Adenine + PRPP becomes AMP + Ppi
• Hypoxanthine-guanine phosphoribosyltransferase also serves in recycling.

Gout

• Related to purine metabolism

Cancer Targets

• Relate to abnormal nucleotide processes

Thymidylate Synthesis

• Thymidylate can be derived from dCDP and dUMP

Summary of Nucleotide Metabolism

• Purine ring system
• Carbamoyl phosphate as the target
• Conversion from Monophosphate to triphosphate
• End products of purine and pyrimidine degradation
• Free purines can be salvaged
• Deficiencies lead to ADA and Lesch-Nyhan syndrome
• The disease, Gout
• Enzymes in the nucleotide biosynthesis pathways as drug targets

Carbohydrate Biosynthesis Overview

• Anabolic pathways are generally reductive.
• Plants are autotrophs.

Plastids

• Most biosynthetic activities of the plant happen in plastids
• Plastids reproduce by binary fission.
• Chloroplasts are the site of CO2 assimilation.
• Chloroplasts can become proplastids, depending on plant tissue type.

Carbon Dioxide Assimilation Stages

• Stage 1: CO2 assimilation, a carbon-fixation stage.
• Stage 2: 3-phosphoglycerate is reduced to form triose phosphates.
• Stage 3: 5/6 triose phosphate is converted to ribulose 1, 5 – bisphosphate (3), the starting material.
• Fructose-6-phosphate is a key intermediate in stage 3 of CO2 assimilation.
• Hexose phosphate can also become pentose bisphosphate.
• Reactions are generally the same in each direction.

Three Stages of C02 Fixation

• Photosynthetic organisms in 1940’s (Calvin) were classified as C3 or C4 plants via incorporation

Rubisco

• Ribulose 1,5 – Bisphosphate Carboxylase is in stage 1
• Rubisco has two distinct forms
• Form I: vascular plants, algae, and cyanobacteria
• Form II: Certain photosynthetic bacteria

Catalytic Mechanism of Rubisco

• Role of Mg2+

Regulation of Rubisco

• Inactive if not carbamoylated on Lys201
• Ribulose 1,5 – bisphosphate inhibits carbamoylation, corrected by Rubisco activase to promotes its release
• Process Regulated by nocturnal inhibitor

Conversion of 3-Phosphoglycerate

• 3- phosphoglycerate formed is converted Glyceraldehyde 3-phosphate in 2 steps, with an exception
• Chloroplast stroma contains all the glycolytic enzymes except phosphoglycerate mutase.
• Reaction includes Stromal 3-phosphoglycerate kinase and Chloroplast specific isozyme: glyceraldehyde 3-phosphate dehydrogenase, and Dihydroxyacetone phosphate (DHAP)

Stage III: Regeneration of Ribulose I,5 Bisphosphate

• Regeneration involves the removal of a 2 carbon ketol group (CH2OH-CO-) for the continuous flow of CO2 into carbohydrate ribulose 1,5 – bisphosphate

Transketolase

• Transketolase catalyzes reactions of the Calvin cycle.
• A general reaction catalyzed by transketolase is shown in the top box
• Middle box – hexose and triose produce 4 and 5 carbon sugars
• Bottom box – conversion of 7 carbon and 3 carbon sugars to two pentoses

Key Cofactor - TPP

• Thiamine Pyrophosphate is a key cofactor

Regulation of C02

• Four regulated enzymes are regulated by light:
  • Ribulose 5 – phosphate kinase
  • Fructose 1,6 – bisphosphatase
  • Sedoheptulose 1,7 – bisphosphatase
  • Glyceraldehyde 3 – phosphate dehydrogenase.

Summary of Photosynthesis

• Photosynthesis in vascular plants takes place in chloroplasts.
• Rubisco condenses CO2 with ribulose 1,5 – bisphosphate and forms an unstable hexose bisphosphate, which splitting into 2 molecules of 3-phosphoglycerate.
• During Stromal isozymes of the glycolytic enzymes catalyzes the reduction of 3 - phosphoglycerate to glyceraldehyde 3 - phosphate
• The stromal enzymes (transketolase, aldolase) rearrange the carbons of triose phosphates
• Fixing three CO 2 into one triose phosphate uses 9 ATP and 6 NADPH.
• Antiporter allows for Pi exchange in the inner chloroplast membrane with the cytosol for 3 – phosphoglycerate or DHAP
• Four enzymes of the Calvin cycle are activated indirectly by light and inactive in the dark

Triose Phosphate

• Movement of triose phosphate moved into cytosol then sucrose and starch accumulation in the chloroplast.
• Function depends whether ATP and NADH can cross the chloroplast membrane or not

Biosynthesis Location

• Production of carbohydrates occurs during bright light.
• Synthesis location sucrose and starch is different from the production location

Glucose Substrate - ADP

• Starch is a high molecular weight polymer of D-glucose.
• There is biosynthesis in chloroplasts for short-term storage, as well as synthesis in the amyloplasts of non-photosynthetic parts.
• A key step is Glucose activation in starch synthesis through an activated nucleotide sugar (ADP-glucose).
• Starch synthase builds Glucose residues from ADP-glucose to preexisting starch molecules.
• Key structural variation for amylose vs. amylopectin

Sucrose Synthesis

• Use UDP-Glucose Substrate
• Most of the triose phosphate in the Calvin cycle becomes sucrose or starch
• Sucrose is synthesized in the cytosol with DHAP and glyceraldehyde 3-phosphate (G3P)

Regulation points - Triose Phosphates

• Regulation involves Triose phosphates regulated at Fructose 1,6 – Bisphosphatase - PPi-dependent phosphofructokinase (PP-PFK-1) also regulates
• Also Fructose 2,6-bisphosphate, also known as F26BP
• Phosphofructokinase-2 is responsible specifically for F26BP, and is inhibited by DHAP and 3-phosphoglycerate, but Stimulated by fructose 6-phosphate and P₁ .
• Sucrose synthesis only occurs if the triose phosphate exceeds what is needed in active reactions of the cycle

Intermediate Pool Elements

• Plant metabolism should be considered as shared intermediate metabolic pools.
• These include Glucose 1-phosphate, glucose 6-phosphate, and fructose 6-phosphate
• And Ribulose, xylulose, ribose
• Also Glyceraldehyde 3-phosphate and DHAP