Lee Jae Yun, Li Zhiqun, Miller Eric S
Department of Plant & Microbial Biology, North Carolina State University, Raleigh, North Carolina, USA.
Department of Plant & Microbial Biology, North Carolina State University, Raleigh, North Carolina, USA
J Bacteriol. 2017 Apr 11;199(9). doi: 10.1128/JB.00855-16. Print 2017 May 1.
The genome of T4-type bacteriophage KVP40 has five genes predicted to encode proteins of pyridine nucleotide metabolism, of which two, and , 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 and expressed in , the proteins were purified, and their enzymatic properties were examined. KVP40 NadV NAmPRTase is active , and a clone complements a mutant defective in both the bacterial 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 reverse transcription-quantitative PCR (qRT-PCR) and enzyme assays of infected cells demonstrated and 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 double-stranded DNA (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. T4-type bacteriophages enhance DNA precursor synthesis through reductive reactions that use NADH/NADPH as the electron donor and NAD for ADP-ribosylation of proteins involved in transcribing and translating the phage genome. We show here that phage KVP40 encodes a functional pyridine nucleotide scavenging pathway that is expressed during the metabolic period of the infection cycle. The pathway is conserved in other large, dsDNA phages in which the two genes, and , share an evolutionary history in their respective phage-host group.
T4型噬菌体KVP40的基因组中有5个基因，预测可编码参与吡啶核苷酸代谢的蛋白质，其中两个基因（nadV和natV）足以构成一条烟酰胺腺嘌呤二核苷酸（NAD）补救途径。NadV是一种明显的烟酰胺磷酸核糖基转移酶（NAmPRTase），而NatV是一种明显的双功能烟酰胺单核苷酸腺苷酸转移酶（NMNATase）和烟酰胺腺嘌呤二核苷酸焦磷酸酶（Nudix水解酶）。编码预测的补救途径的基因被克隆并在大肠杆菌中表达，对蛋白质进行了纯化，并检测了它们的酶学性质。KVP40 NadV NAmPRTase在体外具有活性，并且一个克隆可互补在细菌从头合成途径和补救途径中均有缺陷的大肠杆菌突变体。与其他NAmPRTase类似，KVP40酶表现出ATP酶活性，这表明在反应机制中存在能量偶联。在一个偶联反应系统中测定了NatV NMNATase活性，该系统证明了从烟酰胺、磷酸核糖焦磷酸和ATP生物合成NAD。还表明NatV Nudix水解酶结构域具有活性，其优选底物为ADP-核糖、NAD和NADH。使用逆转录定量PCR（qRT-PCR）进行的表达分析以及对受感染大肠杆菌细胞的酶活性测定表明，在感染的早期和延迟早期，当核苷酸前体代谢的其他KVP40基因表达时，nadV和natV也会转录。NadV和NatV蛋白在几种大型双链DNA（dsDNA）肌尾噬菌体以及一些非常大的长尾噬菌体中的分布和系统发育，表明吡啶核苷酸清除在病毒感染细胞中具有广泛的相关性。NAD生物合成是大型、快速复制的dsDNA噬菌体的另一个重要代谢资源控制点。T4型噬菌体通过还原反应增强DNA前体合成，这些反应使用NADH/NADPH作为电子供体，并使用NAD对参与转录和翻译噬菌体基因组的蛋白质进行ADP-核糖基化。我们在此表明，噬菌体KVP40编码一种功能性的吡啶核苷酸清除途径，该途径在感染周期的代谢期表达。该途径在其他大型dsDNA噬菌体中是保守的，其中nadV和natV这两个基因在它们各自的噬菌体-宿主组中具有共同的进化历史。
