菸醯胺腺嘌呤二核酸補救路徑中菸鹼胺磷酸核糖轉基酶之基因選殖、表達與酵素活性探討

Cloning, Expression and Characterization of Nicotinamide Phosphoribosyltransferase in NAD+ Salvage Pathway

李政洋(Cheng-Yang Li)

指導教授 ： 黃啟清

高雄醫學大學/醫學院/生物化學研究所/碩士(2009年)

https://doi.org/10.6832/KMU.2009.00056

摘要

菸醯胺腺嘌呤二核酸 ( Nicotinamide adenine dinucleotide，NAD+)在生物體內做為能量來源，於正常飲食攝取可由Trptophan自新生合成路徑(de novo pathway)生成，攝取不足情況下NAD+透過補救路徑 (salvage pathway) 以維持體內NAD+/NADH平衡及生理功能。
NAM (nicotinamide)為NAD+代謝後產物之ㄧ，在飲食限制條件下經補救路徑中Nampt(Nicotinamide phosphoribosyltransferase)以NAM為受質反應產生NMN（Nicotinamide mononucleotide）再由Nmnat ( Nicotinamide mononucleotide adenylyltransferase )反應產生NAD+，此反應中Nampt是速率決定酵素。由晶體結構研究中已知Nampt H247位置與一磷酸基產生氫鍵並透過同位素偵測其催化活性已知在ATP環境下酵素效率較高（來源為人類Nampt ；無ATP環境中kcat/Km = (6.1 ± 0.5) × 103 (M-1s-1)，有ATP環境中kcat/Km = (1.8 ± 0.9) × 106 (M-1s-1)）。
為了要了解Nampt在NAD+補救路徑中的活性與H247所扮演的角色，實驗利用老鼠肝臟Nampt cDNA透過PCR增幅後接於經限制酶NdeI與BamHI反應過後之載體pET15b上，再轉型至E. coli DH5α保存，定序確認無誤後轉型至E. Coli BL21（DE3）進行表達，超音波震盪過程中Nampt容易形成包涵體，遂加入0.1% Triton X-100於超音波震盪緩衝液幫助Nampt溶解。在HPLC實驗中，無ATP環境下經過72小時反應物PRPP ( Phosphoribosyl-1-pyrophosphate )並未減少且無NMN生成，但在含有ATP環境下90小時後有NMN生成與ATP裂解後產物出現，此結果與先前對於ATP與Nanmp研究結果相似。由於反應速度很慢並且在UV下偵測訊號很弱，需利用3αHSD/CR催化NAD+還原成NADH以enzyme-coupled fluorometric assay方式偵測。在pH穩定性實驗中發現，Nampt在pH 7.5環境中0~6.5小時內活性不隨時間增加而改變，pH 10環境中0~5小時內活性隨時間增加而減少，於5小時發現蛋白質沉澱，pH 7.5環境下0~3小時內活性並無減少情形，於三小時發現蛋白質沉澱，之後實驗在pH 7.5環境下進行反應，測得Nampt之催化活性，對於受質的KNAM為1.5 ± 0.2 (μM)，V/Et為(3.4 ± 0.1) × 10-2 (s-1)，V/KmEt為(2.2 ± 0.1) × 104 (M-1s-1)，此數據與先前研究相似。以定點突變方法將H247突變為H247A、H247E、H247N及H247S，目的是藉由改變247位置的胺基酸藉以了解H247的功能，目前只有H247S能在超音波震盪上清液收集到，經純化、透析後偵測其酵素活性，H247S對受質的KNAM為0.21 ± 0.03 (μM)，V/Et為(5.7 ± 0.2) × 10-1 (s-1)，V/KmEt為 (2.6 ± 0.1) × 103 (M-1s-1)，與野生型Nampt相比H247S具有較弱的催化活性，可能是由於構型改變或是並無磷酸化現象的發生。

關鍵字

菸醯胺腺嘌呤二核酸 ； 菸鹼胺磷酸核糖轉基酶補救路徑

並列摘要

Nicotinamide adenine dinucleotide (NAD+) is a biological molecule that participates in many metabolic reactions. Basically NAD+ can be synthesis by Trp through de novo pathway, but in carolie restrict environment will be refueled through salvage pathway from different substrate to maintain biological function.
NAM (nicotinamide) is one of the NAD+ metabolic product, during carolie restriction, Nicotinamide phosphoribosyltransferase (Nampt) will catalyze NAM into NMN then NMN will be catalyzed by Nicotinamide mononucleotide adenylyltransferase (Nmnat) form NAD+. In this reaction Nampt is rate-limited enzyme. In earlier researches, Nampt H247 has a hydrogen bond with a phosphate group and has higher activity with ATP (Nampt from human : without ATP kcat/Km = (6.1 ± 0.5) × 103 (M-1s-1) with ATP kcat/Km = (1.8 ± 0.9) × 106 (M-1s-1).
In order to understand the activity of Nampt in NAD+ salvage pathway and the function of H247. Using PCR to amplify Nampt cDNA from mouse liver, ligate with pET15b vector, transform in E. coli DH5α to storage DNA and DNA sequence. After checking DNA sequence correctly, then transform in E. coli BL21 to express Nampt protein. During protein expression, Nampt is easily to form inclusion bodies so we use Triton X-100 to help protein solute in solution. In HPLC experiment, there is no PRPP decrease and NMN synthesis without ATP react after 72 hours reaction but NMN synthesis and ATP degradation product can be found after 90 hours reaction with ATP react. This result is close to earlier research. Because of the signal detected by UV spectrum is weaker than fluorometric spectrum, we use enzyme-coupled fluorometric assay to detect NADH catalyzed from NAD+ through 3αHSD/CR reaction. In protein stability experiment, Nampt is stable in pH 7.5 enviroment from 0~6.5 hours but protein is denatured in pH5.5 and 10 from after 3 and 5 hours incubation. Further experiment will react in pH 7.5 enviroment and KNAM = 1.5 ± 0.2 (μM), V/Et = (3.4 ± 0.1) × 10-2 (s-1) and V/KmEt = (2.2 ± 0.1) × 104 (M-1s-1) this result is consist with earlier research. Then we use site-directed mutagenesis to mutate H247 into Ala, Glu, Asn and Ser to understand the function of H247. H247S KNAM = 0.21 ± 0.03 (μM), V/Et = (5.7 ± 0.2) × 10-1 (s-1) and V/KmEt = (2.6 ± 0.1) × 103 (M-1s-1). Compare with wild type Nampt, wild type Nampt has better catalytic activity, it may be cause by the conformational change or there is no phophorylation.

並列關鍵字

Nicotinamide Phosphoribosyltransferase ； NAD ； Salvage Pathway

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