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PMID:18248423
| Citation |
Bhattacharya, B, Giri, N, Mitra, M and Gupta, SK (2008) Cloning, characterization and expression analysis of nucleotide metabolism-related genes of mycobacteriophage L5. FEMS Microbiol. Lett. 280:64-72 |
|---|---|
| Abstract |
The genomes of mycobacteriophages of the L5 family, which includes the lytic phage D29, contain several genes putatively linked to nucleotide-metabolizing functions. Two such genes, 48 and 50, encoding thymidylate synthase and ribonucleotide reductase (RNR), respectively, were overexpressed in Escherichia coli and the recombinant proteins were biochemically characterized. It was established that Gp50 was a class II RNR having properties similar to that of the corresponding enzyme from Lactobacillus leichmanni, whereas Gp48 was a flavin-dependent thymidylate synthase (ThyX) that resembled the Paramecium bursaria chlorella virus-1 ThyX enzyme in its properties. That both these proteins play a role in phage development was evident from the observation that they were detectable soon after the lytic phase of growth commenced. Gp48 and 50 were also found to coimmunoprecipitate, which indicates the possible existence of an L5 thymidylate synthase complex. Thymidylate synthase assays revealed that during the intracellular stage of phage growth, a significant decrease in the host thymidylate synthase (ThyA) activity occurred. It appears that synthesis of the viral enzyme (ThyX) is necessary to compensate for this loss in activity. In general, the results suggest that phage-encoded nucleotide metabolism-related functions play an important role in the lytic propagation of L5 and related mycobacteriophages. |
| Links |
PubMed Online version:10.1111/j.1574-6968.2007.01047.x |
| Keywords |
Cloning, Molecular; Flavins/metabolism; Lysogeny; Mycobacteriophages/enzymology; Mycobacteriophages/genetics; Mycobacterium smegmatis/metabolism; Mycobacterium smegmatis/virology; Nucleotides/metabolism; Recombinant Proteins/chemistry; Recombinant Proteins/genetics; Recombinant Proteins/metabolism; Ribonucleotide Reductases/chemistry; Ribonucleotide Reductases/genetics; Ribonucleotide Reductases/metabolism; Thymidylate Synthase/chemistry; Thymidylate Synthase/genetics; Thymidylate Synthase/metabolism; Viral Proteins/chemistry; Viral Proteins/genetics; Viral Proteins/metabolism |
Significance
Annotations
| Gene product | Qualifier | GO Term | Evidence Code | with/from | Aspect | Extension | Notes | Status |
|---|---|---|---|---|---|---|---|---|
| GO:0050797: thymidylate synthase (FAD) activity |
ECO:0000314: |
F |
Figure 2b-Release of oxidized FAD confirms that enzyme is a flavoprotein Figure 2c-Observed dUMP-dependent NADPH activity indicates sequential mechanism found within ThyX Figure 2d-Effect of ThyX co-substrate displays function through overlap at NADPH binding site Figure 2e-Conversion of dUMP to dTMP is observed (catalytic activity of ThyX) |
complete | ||||
| GO:0050797: thymidylate synthase (FAD) activity |
ECO:0000314: |
F |
Figure 2 Shows the characterization of gp48 thymidylate synthase (ThyX) activity. Figure 2b shows that it is a flavoprotein because it contains bound FAD. It was also said to be detectable soon after the lytic phase of growth had commenced. |
complete | ||||
| GO:0008998: ribonucleoside-triphosphate reductase activity |
ECO:0000314: |
F |
Figure 3 showed the characterization of ribonucleotide reductase activity of gp50. Figure 3b showed the enzyme could reduce of ATP to dATP. In the presence of various nucleotides, only dGTP stimulated reduction of ATP (Figure 3c). It was stated on the bottom of page 69 at the bottom of the left-hand column, that the enzyme was only found to reduce only triphosphate and not diphosphate. |
complete | ||||
| GO:0008998: ribonucleoside-triphosphate reductase activity |
ECO:0000314: |
F |
Figure 3-Results show the reduction of ATP to dATP with dependence on coenzyme B12 for activity. Dependence confirms Class II reductase. As seen in Figure 3c, only dGTP was found to stimulate reduction. Monitoring of absorbance indicates reduction of only triphosphates. |
complete | ||||
Notes
See also
References
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