The de novo pathway leading to the synthesis of AMP and GMP begins with the transfer of an amido group from glutamine to PRPP (). Since PRPP is used for the both de novo and salvage synthesis of purine and pyrimidine nucleotides as well as for the synthesis of NAD, histidine and tryptophan, any stress that alters PRPP availability affects multiple pathways. Given the essential nature of PRPP, all free-living organisms contain at least one gene encoding PRPP synthetase (PRS; EC 22.214.171.124) (). Recently four PRS cDNAs were isolated from Arabidopsis by functional complementation of an Escherichia coli mutant lacking PRS activity (Genbank accessions X83764 (PRS1), X92974 (PRS2), AJ012406 (PRS3), AJ012407 (PRS4)) (). A fifth PRS sequence that predicts a protein with high amino acid sequence homology to the other Arabidopsis PRSs, particularly with PRS1 and 2 was located on chromosome II and designated PRS5 (). Kinetic characterization of the gene products of PRS1-4 indicated that PRS3 and 4 represent a novel class of PRSs since their activities are independent of inorganic phosphate (Pi). A phylogenetic comparison of 45 putative PRS amino acid sequences from 31 organisms also suggested that PRS3 and 4 are a divergent family, distinct from any of the other PRS sequences (). The subcellular localization of the Arabidopsis PRSs has not yet been determined but may resemble the compartmentation of spinach PRSs that have been localized to the cytosol, chloroplast and mitochondria ().
With all the proper components in place, the engineered de novo pyrimidine pathway was combined with pentose phosphate enzymes and cofactor regeneration of ATP and NADP+ to synthesize UTP from HCO3−, NH4+, aspartate and glucose in one pot. CTP was synthesized from UTP and NH4+ in a separate reaction due to inhibition from components present in the first reaction . shows the overall reaction for synthesis of UTP and CTP. A relatively small number of components are necessary to build the pyrimidine ring. The metabolic origin of each atom is shown in . The major contributor is aspartate, and due to the wide availability of specifically labeled forms of aspartate, many specific pyrimidine base labeled patterns can be synthesized including a combination of 13C, 15N and 2H. The exchange of C6 proton with solvent during the decarboxylation of OMP is convenient for specific deuteration schemes. Deuteration at the C5 position is often desired since the H5 and H1 chemical shifts are very similar, and C5 deuteration has been accomplished previously through a separate chemical synthesis of 2H5- uracil 31. Using the de novo enzymatic synthesis scheme, selective deuteration on both the base and ribose with simultaneous 15N labeling was successful in a single pot reaction.
Nucleotides Metabolism and De Novo Synthesis of Nucleotides
Regeneration of ATP and NADP+, as shown in , provides a driving force for the overall pyrimidine synthesis reaction ,. Several equivalents of creatine phosphate drive the immediate recharging of NMPs and NDPs to NTP by nucleotide kinases through mass action. Similiarly, α-ketoglutarate and NH4+ fuel the conversion of NADPH to NADP+ providing the proper substrate for the NADP+ dependent reactions to move forward. In addition dATP was substituted for ATP in all cases to prevent dilution of the NTP pool with unlabeled ATP. Although de novo pyrimidine enzymes cpkA and pyrG accept dATP with slightly reduced activity, the deoxynucleotide byproducts can be easily separated from the product NTPs during the affinity purification step.
Purines and Pyrimidines Quiz - EHSL
The fourth reaction of de novo pyrimidine biosynthesis is the conversion of DHO to orotate. DODH is thought to catalyze this reaction (). DODH was found to be located on the outer surface of the inner membrane of mitochondria in mammals (). Although there are no detailed studies of a similar enzyme in plants, suggested that tomato DODH is also located in mitochondria.
Purine and Pyrimidine Nucleotide Synthesis and …
Orotate is converted to UMP in two successive reactions catalyzed by orotate phosphoribosyl transferase (OPRT) and orotidine 5′-monophosphate decarboxylase (ODCase). As the intermediate of these steps, orotidine-5′-monophosphate, was not detected in plant tissue, and these two activities co-purified through several purification steps, it was suggested that these two enzymes form a complex in vivo (; ). In fact, recent results demonstrate that OPRT and ODCase reside in a single polypeptide that is termed UMP synthase. This bifunctional enzyme catalyzes the last two steps of the de novo pyrimidine pathway in plants as well as mammals (). This structure improves the efficiency of these reactions by channeling the product of the first reaction to the second enzyme without dissociation from the complex. In most organisms, except some parasitic protozoans, the N-terminal portion of this bifunctional enzyme has sequence identity with OPRT while the C-terminal region has identity with ODCase (, , , ). In some parasitic protozoans the order of the activities within the enzyme is reversed () suggesting that a bifunctional UMPS has arisen more than once during the course of evolution.