Unnatural Base Pairs
De novo Synthesis of Purine and Pyrimidine Bases
De novo Synthesis of Pyrimidine Bases
The de novo synthesis of pyrimidines starts with the assembly of the orotate ring which is then converted into a pyrimidine nucleotide by binding the ring to a ribose phosphate (Berg et al., 2012). The first step in the synthesis of the pyrimidine ring is the formation of carbamoyl phosphate, which is formed from hydrogencarbonate and ammonia. The reaction is catalyzed by carbamoyl phosphate synthase (CPS) and requires two molecules of ATP. Glutamine is the main source for ammonia, which is produced by the hydrolysis of glutamine. This reaction is also catalyzed by CPS and yields ammonia and glutamate. Carbamoyl phosphate is converted into carbamoyl aspartate by aspartate carbamoyltransferase (ACT) through a reaction with aspartate. In turn, carbamoyl aspartate is oxidized to orotate, closing the ring structure. 5-phosphoribosyl-1-pyrophosphate (PRPP) reacts with orotate, a reaction that is catalyzed by orotate phosphoribosyltransferase (OPRTase). This reaction yields orotidylat, which in the next step is decarboxylated by oridylate decarboxylase (OCD) to uridylate (UMP). UMP acts as a precursor for the synthesis of cytidine. The first step in the synthesis of cytidine from UMP involves the phosphorylation of UMP to UTP . This reaction involves two steps. In the first step, UMP is converted to UDP by a specific nucleoside monophosphate kinase, the UMP kinase. ATP acts as a phosphate donor and is converted to ADP. UDP can now be converted to UTP by nucleoside diphosphate kinases, which are relatively unspecific. In the second step, UTP is converted to CTP in a reaction catalyzed by the cytidine triphosphate synthetase through the exchange of a carbonyl group with an amino group. CTP is subsequently converted into dCTP, a reaction that is catalyzed by ribonucleoside-triphosphate reductases (RTPR). RTPR also catalyzes the reaction of UTP to dUTP, which is then converted to dTMP through three consecutive reactions. dTMP is phosphorylated by dTMP kinases, yielding dTDP, which is then phosphorylated by nucleoside diphosphate kinases to dTTP.
De novo Synthesis of Purine Bases
Purine bases are produced de novo directly on the ribose (Berg et al., 2012). The synthesis starts with the replacement of the pyrophosphate of PRPP with an amino group, yielding phosphoribosylamine (PRA). This reaction is catalyzed by amidophosphoribosyltransferase (ATase) and also uses the ammonia from a glutamine side-chain as the donor of the amino group. The conversion of PRPP to PRA is a committing step in the purine biosynthesis. The synthesis of the purine ring involves nine additional steps with the first six reactions being relatively similar. In every reaction, an oxygen atom which is bound to a carbon atom is activated by phosphorylation and a subsequent substitution by ammonia or an amino-group, which act as a nucleophile agent. These subsequent reactions lead to the formation of inosinate (IMP), which acts as a key intermediate in the purine synthesis. Inosinate is converted into either AMP or GMP. AMP is synthesized by a substitution of the C-6 carbonyl oxygen with an amino group by adenylosuccinate synthase (ASS). In this reaction, GTP instead of ATP is used as a donor of the phosphoryl group. The conversion of IMP to GMP is catalyzed by the GMP synthase and starts with the oxidation of IMP to xanthylate (XMP) and the subsequent addition of an amino group. In a second step, XMP is converted into GMP, a reaction that requires ATP as a donor for an AMP group. GMP and AMP are again phosphorylated to GTP and ATP by specific kinases.
Conversion of Ribonucleosid Diphosphates to Deoxyribonucleotides
Deoxyribonucleotides are synthesized from ribonucleotides by substitution of the 2´-hydroxyl group of the ribose by a hydrogen. The reaction is catalyzed by the enzyme ribonucleotide reductase, which is strongly conserved in all living organisms (Berg et al., 2012). In E. coli, two main types of ribonucleotide reductases exist. Ribonucleoside-triphosphate reductases can convert ribonucleoside-triphosphates into deoxyribonucleoside-triphosphates, while ribonucleoside-diphosphate reductases convert ribonucleoside-diphosphates to deoxyribonucleoside-diphosphates (Kanehisa and Goto, 2000).
Salvage Pathways
Both purine and pyrimidine bases can be recycled and converted into the corresponding nucleotides through salvage pathways. Adenine can be recycled through conversion into AMP, a reaction that is catalyzed by the adenine phosphoribosyltransferase and requires PRPP . AMP can then be subsequently converted into ATP or dATP as described above. Hypoxanthine guanine phosphoribosyltransferase (HGPRT) catalyzes the recycling of guanosine, a reaction that also requires PRPP as a donor for a phosphate. HGPRT also catalyzes the conversion of hypoxanthine to IMP which again is a precursor of GMP and AMP. The recycling of thymine involves two steps: in the first step, thymine is converted to thymidine by the thymidine phosphorylase. In a second step, thymidine is converted to TMP by thymidine kinase. Cytosine can be recycled by conversion to uracil, a reaction that is catalyzed by cytosine deaminase. Following the conversion to UTP, CTP is produced by CTP synthase. The recycling of bases saves intracellular energy, since the de novo synthesis requires large amounts of ATP. Therefore, the recycling of bases through salvage pathways is usually favored by cells.
