[目的]为研究短链壬基酚聚氧乙烯醚脱氢酶(sNPEO-DH)的脱氢氧化机制(基因克隆于Ensifer sp.AS08),我们进行了以下实验.[方法]采用同源序列比对及同源建模的方法筛选出与其辅酶黄素腺嘌呤二核苷酸(FAD)异咯嗪基邻近的4个氨基酸残基.以定点突变方法分别构建了突变体,并进行了重组蛋白的表达纯化和酶活力测定.[结果]野生型和突变体的酶学动力学实验表明,突变体N90A和N509A对亲水性底物聚乙二醇(PEG1000)的相对活性分别降低为51%和89%,对疏水性底物sNPEO的活性分别降低为26%和40%,说明氨基酸残基N90和N509可能与底物的结合相关.突变体H465A的相对活性丧失了90%以上,突变体N507A完全丧失活性;瞬时"停-流"检测实验进一步证明N507A突变体阻断了底物向FAD传递质子的过程,突变体H465A阻断了对FAD还原形成的FADHZ脱氢再生的过程.[结论]以上结果说明N507和H465为sNPEO脱氢酶活性中心中参与对底物氧化脱氢及FADH2脱氢再生进行下一次反应的催化位点.%[Objective]To study dehydrogenation by short ethoxy chain nonylphenols dehydrogenase (sNPEO-DH) from Ensifer sp.AS08.[Methods]We screened four amino acid residues of sNPEO-DH that are adjacent to the isoalloxazine ring of the coenzyme flavin adenine dinucleotide ( FAD ) by using multiple sequence alignment and homology modeling.Mutations were introduced by site-directed mutagenesis.The recombinant proteins were expressed, purified and assayed.[Results]The relative activities of mutants N90A and N509A against the hydrophilic substrate PEG1000 decreased to 51％ and 89％ , respectively, and against the hydrophobic substrate NPEOav10 decreased to 26％ and 40％ , respectively; indicating that N90 and N509 might be related to substrate binding.The relative activity of mutant H465A and N507A lost 90％ and 100％ , respectively; "stop-flow" experiments revealed that the processes of proton transfer from substrate to FAD and from FAD to enzyme were blocked in mutant N507A and H465A, respectively.[Conclusion]Amino acid residues N507 and H465 located at the activity center of sNPEO-DH and play roles as catalytic sites for the oxidative dehydrogenation of the substrates and FADH2, respectively.
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