Properties of NadV / NatV Proteins in a Pyridine Nucleotide (NAD+) Scavenging System Encoded by Vibriophage KVP40

adenylyltransferase;NAD;Bacteriophage KVP40;NAmPRTase;NadV;NatV

Li, Zhiqun ; Eric S. Miller, Committee Chair,Li, Zhiqun ; Eric S. Miller ; Committee Chair

University:North Carolina State University

关键词: adenylyltransferase;    NAD;    Bacteriophage KVP40;    NAmPRTase;    NadV;    NatV;   

Others  :  https://repository.lib.ncsu.edu/bitstream/handle/1840.16/1990/etd.pdf?sequence=1&isAllowed=y

美国|英语

来源: null

【 摘 要 】

Bacteriophage KVP40 is a T4-type phage that infects Vibrio species. In the phage 245 kbp dsDNA genome, a set of bacterial-like genes, nadV, natV, pnuC, nadR and sir2 are predicted to be involved in the synthesis and recycling of nicotinamide adenine dinucleotide (NAD+). Comparison with proteins of known function indicates that NadV possesses a nicotinamide phosphoribosyl-transferase domain (NAmPRTase) that converts nicotinamide (NAm) to nicotinamide mononucleotide (NMN). NatV has two predicted functional domains, where the N-terminal region is a NMN adenylyltransferase (NMN ATase) converting NMN to NAD+, and the C-terminal region is a nudix hydrolase. NadR / PnuC and Sir2 are predicted to be NAm / NMN membrane transporters and to degrade NAD+, repectively. The purpose of this project was to biochemically characterize the properties of the KVP40 NadV / NatV proteins, two critical enzymes in the hypothetical NAD+ scavenging system. nadV and natV had been cloned, purified, and constructed plasmids nadV pZL405, natV pZL176 and pZL166. Sequence of nadV in pZL405 had been confirmed as wild type. Complementation experiment transformed pZL405 into Salmonella typhimurium auxotrophs restored its NAD+-independent growth, indicating the nadV of KVP40 is a functional gene. NadV / NatV were expressed using BL21-AI expression system and purified using Ni-NTA technique. Sequences of purified NadV / NatV were confirmed as wild type using LC-MASS Spectrometry, and sent for antibodies preparations. Western blot confirmed anti-NadV serum positively interacted with NadV samples. Furthermore, NadV enzyme assay was also performed, analyzed using HPLC method and confirmed the NAmPRTase function. An interesting phenomenon was discovered during the enzyme assay, that the NAmPRTase function of KVP40 NadV could be carried out with and without ATP added, indicated NadV of KVP40 also possesses an ATPase function, and the kinetics of the reaction catalyzed by NAmPRTase of NadV possibly affected by ATP.