The Deoxycytidine Pathway for Thymidylate Synthesis in Escherichia coli

Abstract

When thymidylate production is diminished by a mutation affecting dCTP deaminase, Escherichia coli is known to use an alternate pathway involving deoxycytidine as an intermediate. The pathway requires the gene for any of three nucleoside diphosphate kinases (ndk, pykA, or pykF) and the gene for a 5-nucleotidase (yfbR).Escherichia coli has three known routes for the aerobic de novo synthesis of dUMP, the substrate for thymidylate synthetase. They are referred to as the dCTP, dUDP, and deoxycytidine (dCyd) (17, 29) pathways after their characteristic intermediates. In E. coli and in Salmonella enterica serovar Typhimurium, dCTP is a precursor for 75% to 80% of thymidylate that is synthesized de novo (17). In the first step of this dCTP pathway, dCTP is converted to dUTP by dCTP deaminase, which is encoded by the dcd gene (Fig. 1). Then, dUTPase (dut gene) catalyzes the hydrolysis of a pyrophosphate from dUTP, yielding dUMP. Most of the remaining synthesis of dUMP (and hence TMP) is presumed to occur through the dUDP pathway, in which UDP is reduced enzymatically to dUDP. Some of the dUDP may produce dUMP through the reversible thymidylate kinase reaction, but most of it is phosphorylated to dUTP, which is then degraded to dUMP by dUTPase (Fig. 1). The dCyd pathway (Fig. 2) comes into play when dCTP deaminase is inactivated by a dcd mutation, thereby disrupting the major (dCTP) pathway for dUMP synthesis (29). dcd mutants grow poorly in the absence of thymidine (Thd), indicating that the dUDP pathway is inadequate to supply the needs of the cell. However, dcd mutants readily acquire Thd independence through spontaneous mutations in the deoA gene, which encodes a phosphorylase for both Thd and deoxyuridine (dUrd) (29). This finding suggested that dUrd could be an endogenous precursor for TMP. It was surprising because de novo synthesis of nucleic acid precursors occurs through nucleotide rather than nucleoside intermediates. Therefore, the dCyd pathway must rely on salvage enzymes. The blockage of dCTP deaminase in dcd mutants was postulated to lead to a metabolic detour. The massively accumulating dCTP is degraded to dCyd, which is then deaminated to dUrd. dUrd is converted to dUMP by Thd kinase, which, like other enzymes that act on Thd, acts on dUrd as well. The deoA mutation enables this pathway by blocking the breakdown of a critical intermediate, dUrd, and thereby renders a dcd mutant Thd independent. The major evidence for this pathway was that a mutation in cdd, the gene for cytidine (and dCyd) deaminase, caused a dcd deoA mutant to again require Thd (29), indicating that dCyd and dUrd are intermediates. In this paper, a similar approach is used to identify the additional enzymes that are critical for this pathway, those involved in each step of the conversion of dCTP to dCyd. The disruption of the corresponding gene should cause a dcd deoA mutant to require Thd.Strain construction. A series of mutations that might affect the dCyd pathway (Table 1) were transduced into BW1929 (dcd deoA) for testing. The pykF...

Structural Insight into the Mechanism of Substrate Specificity and Catalytic Activity of an HD-Domain Phosphohydrolase: The 5′-Deoxyribonucleotidase YfbR from Escherichia coli

“…These results provide further insight into the function of YfbR, an essential component of the deoxycytidine nucleotide scavenging pathway for thymidylate synthesis in E. coli. 10 The substrate binding mode demonstrated here differs significantly from those demonstrated and proposed for other members of the HD-domain phosphohydrolase superfamily. The known substrates of the HD-domain phosphohydrolases include such various nucleotides as 5′-and 2′-deoxyribo-and ribonucleoside monophosphates, (p)ppGpp, and (d)NMPs.…”

“…6 YfbR has been established as an essential component of the deoxycytidine pathway for de novo synthesis of thymidylate in E. coli. 10 The human genome encodes one YfbR ortholog, the uncharacterized HD-domain-containing protein HDDC2 (28% sequence identity), which might represent a novel intracellular 5′-NT in humans. In addition to YfbR, the E. coli genome encodes two more stand-alone HD-domain proteins: YfdR (178 amino acids) and YedJ (231 amino acids).…”

The phtC-phtD Locus Equips Legionella pneumophila for Thymidine Salvage and Replication in Macrophages

“…In E. coli, dTMP synthesis via the deoxycytidine (dCyd) pathway recruits several salvage functions to generate the dUMP substrate for ThyA (46,54). Together, our data indicate that the PhtC and PhtD proteins may be part of a salvage pathway that could be integrated with de novo routes to modulate nucleotide metabolism in L. pneumophila.…”

Thymineless Death Lives On: New Insights into a Classic Phenomenon

“…In the absence of the dCTP deaminase, E. coli can readily adopt another pathway for dUMP production that relies on deoxycytidine deamination (Supplement Figure 1a) (121). That pathway utilizes a 5 deoxynucleotide phosphatase, YfbR, one of three nucleotidases identified in a general screen (98).…”