KVP40 is a T4-like bacteriophage whose dsDNA genome (244,835 bp) has been sequenced (21). It infects Vibrio parahaemolyticus, which can cause disease in fish and shellfish, and gastroenteritis in humans when consumed in raw or under-cooked seafood (i.e., oysters). The KVP40 genome carries bacterial-like genes that have the potential to encode a pyridine nucleotide scavenging system for synthesis of NAD+. This is the first pyridine nucleotide salvage pathway predicted from a phage or eukaryotic viral genome (21). nadV and natV are the two genes that encode the hypothetical two-reaction NAD+ scavenging pathway. NadV, a nicotinamide phosphoribosyltransferase, catalyzes the first reaction that converts nicotinamide to nicotinamide mononucleotide (NMN). The NatV NMN adenylyltransferase activity yields NAD+. Nicotinamide adenine dinucleotide (NAD+) is an essential cofactor involved in fundamental processes in cell metabolism, and pathways for its synthesis are potential anti-microbial, therapeutic targets. The phage enzymes provide model targets for these studies. NatV, which catalyzes the second step of the pathway, also has a Nudix hydrolase domain in the C-terminal half. The purpose of this project was to characterize expression patterns of nadV and natV during KVP40 development in V. parahaemolyticus by using qRT-PCR; to characterize the enzymatic activity of NadV and NatV in a coupled-enzyme assay using alcohol dehydrogenase (ADH) to connect to fluorescent NADH; to identify possible substrates for the NatV Nudix hydrolase; and to use mass spectrometry to quantify product yields of the NatV reactions. qRT-PCR analysis showed that KVP40 nadV and natV were expressed early during infection relative to other phage genes used in this study. NadV-His6 and NatV-His6 enzymes were successfully purified by Ni2+ affinity HPLC and the levels of NADH produced were measured in a three step NadV-NatV-ADH reaction system. In the coupled assay, NatV-His6 converted NMN to 50 μmole of NAD+/sec/μg at 25°C. NatV NMNATase activity was also confirmed by mass spectrometry, in this assay, the rate was 350 μmole NAD+/sec/μg NatV-His6 at 25°C. NadV NAmPRTase activity was confirmed by producing 5.2, 4.9 and 5.0 μmole NMN/sec/μg NadV-His6 enzyme at 25, 30 and 37°C, respectively. Purified NadV used in the coupled enzyme also produced 2.7, 1.5, 1.3, 1.8 or 1.5 μmole NMN/sec/μg NadV-His6, when 10, 25, 40, 50 or 100 pmole NadV-His6 was respectively used in the reaction at 25°C. The âNudixâ activity of purified NatV was measured by a phosphate releasing assay and by mass spectrometry. Using the phosphate release Nudix hydrolase assay, ADP-ribose was identified as a preferred substrate for KVP40 NatV, followed by NAD+, NADH, and NADPH. In this assay, ADP-ribose as substrate yielded 0.6 μmole phosphate/sec/μg NatV-His6 at 37°C, when Mg2+ was supplied as the required metal ion. Mass spectrometry verified the NatV Nudix hydrolase preference for ADP-ribose as substrate, yielding the same rate of 0.6 μmole AMP/sec/μg NatV-His6 at 37°C in the presence of Mg2+. Together these data confirm the various enzymatic activities of key pyridine nucleotide scavenging enzymes encoded by phage KVP40. The time course of expression and in vivo activities suggest that pyridine nucleotide scavenging during KVP40 infection of V. parahaemolyticus is a functional and potential relevant pathway used by the phage.
| Identifer | oai:union.ndltd.org:NCSU/oai:NCSU:etd-07312008-124105 |
| Date | 26 August 2008 |
| Creators | Lee, Jae Yun |
| Contributors | Eric Miller, Paul Orndorff, Jon Olson, Amy Grunden |
| Publisher | NCSU |
| Source Sets | North Carolina State University |
| Language | English |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | http://www.lib.ncsu.edu/theses/available/etd-07312008-124105/ |
| Rights | unrestricted, I hereby certify that, if appropriate, I have obtained and attached hereto a written permission statement from the owner(s) of each third party copyrighted matter to be included in my thesis, dis sertation, or project report, allowing distribution as specified below. I certify that the version I submitted is the same as that approved by my advisory committee. I hereby grant to NC State University or its agents the non-exclusive license to archive and make accessible, under the conditions specified below, my thesis, dissertation, or project report in whole or in part in all forms of media, now or hereafter known. I retain all other ownership rights to the copyright of the thesis, dissertation or project report. I also retain the right to use in future works (such as articles or books) all or part of this thesis, dissertation, or project report. |
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