长双歧杆菌N-乙酰氨基己糖1-位激酶的活性位点

Active sites of N-acetylhexosamine 1-kinase from Bifidobacterium longum

摘要:

【目的】研究长双歧杆菌(Bifidobacterium longum) JCM1217的N-乙酰氨基己糖1-位激酶(N-acetylhexosamine 1-kinase, NahK)中对催化活性有影响的位点。【方法】利用点突变试剂盒,获得NahK的4个位点的共10种单点突变体表达菌株。诱导表达并纯化野生型和突变体酶,用DNS法和NADH偶联的微孔板分光光度法检测野生型及突变体酶的最适pH和最适Mg²⁺浓度,并测定酶促反应动力学参数。【结果】 D208A、D208N、D208E和I24A四种突变体的催化活性几乎丧失。突变体H31A、H31V、F247A和I24V的最适pH由野生型的7.5变为7.0,突变体H31A和F247A的最适Mg²⁺浓度由野生型的5 mmol/L变为10 mmol/L。反应动力学参数测定结果表明,突变体F247Y对底物GlcNAc/GalNAc及ATP的催化活性均高于野生型。【结论】通过定点突变,确定了对NahK催化活性有影响的4个位点,并且获得了一个催化效率提高的突变体(F247Y),为进一步对NahK进行分子改造奠定了一定基础。

Abstract:

[Objective] To study the active sites of N-acetylhexosamine 1-kinase(NahK) from Bifidobacterium longum JCM1217.[Methods] We obtained expression strains of 10 single-mutants at 4 sites of NahK by site-directed mutagenesis, and expressed and purified both wild-type(WT) and mutant enzymes. Then, their optimum pH and optimum concentration of Mg²⁺ were determined by DNS assay and NADH-coupled microplate photometric assay, and their kinetic parameters were measured.[Results] Four mutants(D208A, D208N, D208E and I24A) lost most part of the catalytic activity. The optimum pH of mutants H31A, H31V, F247A and I24V switched from pH 7.5(for WT) to pH 7.0, and the optimum concentration of Mg²⁺ of mutants H31A and F247A increased to 10 mmol/L from 5 mmol/L(for WT). The kinetic parameters of WT and mutants indicate that mutant F247Y had higher enzymatic activity toward GlcNAc, GalNAc and ATP than WT.[Conclusion] The key amino acids that affect the catalytic activity of NahK were determined by site-directed mutagenesis, and together with the mutant that has higher catalytic efficiency, this has laid a foundation for further modification and evolution of NahK.