nucleotide; biosynthesis; purine; pyrimidine; multifunctional protein

For cells that don’t excrete deoxyribonucleosides but instead degrade them further, that further degradation usually involves attack by nucleoside phosphorylases to yield deoxyribose-1-phosphate plus the free base. Further degradation of the purine and pyrimidine bases proceeds by pathways outlined in Purine ribonucleotide metabolism and Pyrimidine ribonucleotide metabolism.

T1 - Pathway engineered enzymatic de novo purine nucleotide synthesis

The biosynthesis of purine and pyrimidine nucleotides takes place over synthetic pathways from small molecules and by salvage pathways from preformed purine or pyrimidine bases or nucleosides. The pathways of synthesis are the same in animals and microorganisms. Salvage pathways are considerably more energy‐efficient than pathways, which require 5 (pyrimidine) or 6 (purine) moles of ATP for each mole of nucleotide produced. Salvage pathways are integral to the cause or treatment of a number of human diseases of purine or pyrimidine metabolism. Among disorders of purine metabolism, the Lesch–Nyhan disease is characterised by overproduction of uric acid, clinical gout, nephropathy, neurologic disease, and unusual self‐injurious behaviours.


Synthesis of Purine Ribonucleotides

Synthesis of Pyrimidine Ribonucleotides

Selective incorporation of isotope labels into nucleotides can be effected by chemical and/or enzymatic synthesis. Using 13C,2H-labeled glucose and unlabeled bases, efficient enzymatic synthesis of a variety of specific ribose labeled nucleotides was carried out, facilitating the study of RNA molecules of increasing size (–, ). Specific labeling of the ribose moiety permits assignment of sugar-sugar and intermolecular NOE correlations by reducing ambiguity and crowding of the spectra. The two most important protons for NMR structural analysis, H1′ and H2′, can be selectively observed, and the variety of available labeling patterns for the glucose starting material provides a great degree of flexibility for labeling of the ribose moiety ().


to IMP in keeping with the total need for purine nucleotide synthesis

Regulation of the rate of pyrimidine nucleotide synthesis in bacteria occurs inlarge part through aspartate transcarbamoylase (ATCase), which catalyzes thefirst reaction in the sequence and is inhibited by CTP, the end product of thesequence (Fig. 16–4a). The bacterial ATCase molecule consists of six catalyticsubunits and six regulatory subunits. The catalytic subunits bind the substratemolecules, and the allosteric subunits bind the allosteric inhibitor, CTP. Theentire ATCase molecule, as well as its subunits, exists in two conformations,active and inactive. When CTP is not bound to the regulatory subunits, theenzyme is maximally active. As CTP accumulates and binds to the regulatorysubunits, they undergo a change in conformation. This change is transmitted tothe catalytic subunits, which then also shift to an inactive conformation. ATPprevents the changes induced by CTP.

Pathway Engineered Enzymatic de Novo Purine Nucleotide Synthesis.

A general method for isotopic labeling of the purine base moiety of nucleotides and RNA has been developed through biochemical pathway engineering in vitro. A synthetic scheme was designed and implemented utilizing recombinant enzymes from the pentose phosphate and de novo purine synthesis pathways, with regeneration of folate, aspartate, glutamine, ATP, and NADPH cofactors, in a single-pot reaction. Syntheses proceeded quickly and efficiently in comparison to chemical methods with isolated yields up to 66% for 13C-, 15N-enriched ATP and GTP. The scheme is robust and flexible, requiring only serine, NH4+, glucose, and CO2 as stoichiometric precursors in labeled form. Using this approach, U- 13C-GTP, U-13C,15N- GTP, 13C 2,8- ATP, and U-15N- GTP were synthesized on a millimole scale, and the utility of the isotope labeling is illustrated in NMR spectra of HIV-2 transactivation region RNA containing 13C2,8- adenosine and 15N1,3,7,9,2-guanosine. Pathway engineering in vitro permits complex synthetic cascades to be effected, expanding the applicability of enzymatic synthesis.

Nucleotides: Their Synthesis and Degradation ..

A general method for isotopic labeling of the purine base moiety of nucleotides and RNA has been developed through biochemical pathway engineering . A synthetic scheme was designed and implemented utilizing recombinant enzymes from the pentose phosphate and purine synthesis pathways, with regeneration of folate, aspartate, glutamine, ATP, and NADPH cofactors, in a single-pot reaction. Syntheses proceeded quickly and efficiently in comparison to chemical methods with isolated yields up to 66% for 13C-, 15N-enriched ATP and GTP. The scheme is robust and flexible, requiring only serine, NH4+, glucose, and CO2 as stoichiometric precursors in labeled form. Using this approach, U-13C- GTP, U-13C,15N- GTP, 13C2,8- ATP, and U-15N- GTP were synthesized on a millimole scale, and the utility of the isotope labeling is illustrated in NMR spectra of HIV-2 transactivation region RNA containing 13C2,8-adenosine and 15N1,3,7,9,2-guanosine. Pathway engineering permits complex synthetic cascades to be effected, expanding the applicability of enzymatic synthesis.