基于理性设计的烟酰胺核糖激酶活性提升及应用

Improving the activity of nicotinamide riboside kinase by rational design and application

烟酰胺单核苷酸(nicotinamide mononucleotide,NMN)是NAD+的前体,补充NMN可有效恢复体内NAD+水平,并改善与NAD+下降相关的病理状态和疾病.目前NMN主要通过酶法生产,但是酶的催化活性较低,使得酶法合成NMN的成本仍然较高.来源于人的烟酰胺核糖激酶NRK1可以催化ATP和烟酰胺核糖合成NMN,因此,以提升烟酰胺核糖激酶NRK1的催化活性为目的,利用理性设计的策略和定点突变技术,通过氨基酸序列比对和结构分析,设计了NRK1的2个突变体NRK1(T47V)和NRK1(N62D),并在大肠杆菌中成功表达.实验表征发现,二者的催化特性均得到了提升,且NRK1(T47V)具有更好的催化特性,其活性较NRK1提高130%,催化效率k_(cat)/K_(m)和稳定性分别较NRK1提高了100%和20%.在较大反应体系中反应100 min时,NRK1(T47V)催化底物烟酰胺核糖的转化率为78%,较NRK1提高15.4%,产物NMN的产量为4.13 g/L,较NRK1提高38%.该结果表明,活性中心附近氨基酸的理性突变能显著提高NRK1酶的催化活性.

Nicotinamide mononucleotide(NMN)is the precursor of NAD+,and the supplement of NMN can restore NAD+levels in vivo,which can alleviatethe pathological conditions and diseases associated with NAD+decline.At present,NMN is mainly produced by enzymatic method,but the catalytic activity of enzyme is still very low,which leads to the higher cost of enzymatically synthesizing NMN.Nicotinamide riboside kinase NRK1 from human can catalyze ATP and nicotinamide riboside synthesizing NMN.Therefore,in order to enhance the catalytic activity of nicotinamide riboside kinase NRK1,two mutants,NRK1(T47V)and NRK1(N62D),were designed based on amino acid sequence alignment and structural analysis,and successfully expressed in Escherichia coli by rational design strategy and site-directed mutagenesis.The experimental results showed that the catalytic characteristics of both of them were improved,and NRK1(T47V)had better catalytic characteristics,whose catalytic activity was 130%higher than NRK,and the catalytic efficiency k _(cat)/K _(m) and stability were 100%and 20%higher than NRK1,respectively.After reaction for 100 min in a bigger reaction system,the conversion ratio of substrate nicotinamide ribose catalyzed by NRK1(T47V)was 78%,was 15.4%higher than that of NRK1,and the yield of NMN was 4.13 g/L,was 38%higher than that of NRK1.These results indicated that rational mutation of amino acids near the active sites could significantly increase the catalytic activity of NRK1 enzyme.