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PMID:28167526

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Citation

'Lee, JY, Li, Z and Miller, ES (2017) Vibrio phage KVP40 Encodes a Functional NAD+ Salvage Pathway. J. Bacteriol. '

Abstract

The genome of T4-type Vibrio bacteriophage KVP40 has five genes predicted to encode proteins of pyridine nucleotide metabolism, of which two, nadV and natV, would suffice for an NAD(+) salvage pathway. NadV is an apparent nicotinamide phosphoribosyltransferase (NAmPRTase) and NatV is an apparent bifunctional nicotinamide mononucleotide adenylyltransferase (NMNATase) and nicotinamide-adenine dinucleotide pyrophosphatase (Nudix hydrolase). Genes encoding the predicted salvage pathway were cloned, expressed in E. coli, the proteins purified and their enzymatic properties examined. KVP40 NadV NAmPRTase is active in vitro and a clone complements a Salmonella mutant defective in both the bacterial de novo and salvage pathways. Similar to other NAmPRTases, the KVP40 enzyme displayed ATPase activity indicative of energy coupling in the reaction mechanism. The NatV NMNATase activity was measured in a coupled reaction system demonstrating NAD(+) biosynthesis from nicotinamide, phosphoribosyl pyrophosphate and ATP. The NatV Nudix hydrolase domain was also shown to be active, with preferred substrates of ADP-ribose, NAD(+) and NADH. Expression analysis using qRT-PCR and enzyme assays of infected V. parahaemolyticus cells demonstrated nadV and natV transcription during the early and delayed-early periods of infection when other KVP40 genes of nucleotide precursor metabolism are expressed. The distribution and phylogeny of NadV and NatV proteins among several large dsDNA myophages, and also those from some very large siphophages, suggest broad relevance of pyridine nucleotide scavenging in virus infected cells. NAD(+) biosynthesis presents another important metabolic resource control point by large, rapidly replicating dsDNA bacteriophages.

Links

PubMed Online version:10.1128/JB.00855-16

Keywords


Significance

Annotations

Gene product Qualifier GO Term Evidence Code with/from Aspect Extension Notes Status

BPKVM:Q6WHZ2

GO:0000309: nicotinamide-nucleotide adenylyltransferase activity

ECO:0000314:

F

Figure 7 shows substrate specificity of NatV Nudix hydrolase as measured by Pi released from the respective substrate. At 37 degree centigrade, the preferred substrate are ADP-ribose, NAD+ and NADH with little differences in activity between them. The stoichiometry of Pi from all of these Nudix hydrolase reactions is 1:1, most yielding an AMP. In reactions where AMP could be directly measured using mass spectrophotometer, ADP-ribose was also preferred substrate, with NAD+, NADH, with FAD showing reactivity but not at a lower rate

complete
CACAO 12595

BPKVM:Q6WHP0

GO:0009435: NAD biosynthetic process

ECO:0000314:

P

Figure 3 and 5show ATP activity and complementation of Samonella Typhimurium pyridine nucleotide auxotroph by the nadV+clone (pZL405nadV+) Figure 7 (explanation from the manuscript).

Figure Title: Substrate specificity of the KVP40 NatV Nudix pyrophosphatase.

A) Activity was measured in the phosphate release assay using 1.5 mM of substrate and 50 pmole NatV-His6 present in reactions incubated at 37°C. Values shown are averages from two assays. Stoichiometry of phosphate per substrate is 1:1.


B) Mass spectrometry analysis of KVP40 NatV 754 Nudix hydrolase substrate specificity. Specific activity was obtained with 1.5 mM substrate and 1 μg NatV, incubated at 37°C. Values are averages from duplicate assays (triplicate for ADP-ribose). The specific activity on ADP-ribose was 0.6 μmole AMP sec^-1 µg^-1 NatV-His6.

complete
CACAO 12596

Notes

See also

References

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