家蚕吡哆醛激酶基因定点突变及突变体功能

Site-directed mutation of pyridoxal kinase gene and the function of the mutants in the silkworm,Bombyx mori

【目的】吡哆醛激酶(pyridoxal kinase,PLK)是维生素B6关键代谢酶。前期研究克隆出家蚕Bombyx mori吡哆醛激酶cDNA,经序列比对发现几个重要且保守的氨基酸残基在此蛋白中被替换。为明确家蚕吡哆醛激酶分子若干特定位置上氨基酸残基在酶功能上的作用进行本研究。【方法】采用重叠延伸法对家蚕吡哆醛激酶Thr47,Asn121,Ile54,Arg88和Trp230氨基酸残基进行定点突变,构建表达载体pET-22b(+)-PLK并转入大肠杆菌Escherichia coli Rosetta中进行诱导表达,经亲和层析对重组蛋白进行纯化,通过酶活性检测进行功能鉴定。【结果】家蚕吡哆醛激酶Thr47,Ile54和Arg88氨基酸突变后酶的催化活力分别下降82%,58%和85%;Asn121突变对酶的催化活力几乎没有影响;而Trp230突变导致酶丧失催化活性。【结论】本研究明确了选定氨基酸侧链基团在家蚕吡哆醛激酶催化功能上的意义。

[Aim] Pyridoxal kinase（PLK） is a key enzyme related to VB6 metabolism.In the previous study,the cDNA of PLK of the silkworm,Bombyx mori,was cloned,and several important and conservative amino acid residues were replaced in this protein by sequence alignment.This study aims to identify the effect of amino acid residues at specific position of PLK of B.mori on enzyme function.[Methods] The conserved sites Thr47,Asn121,Ile54,Arg88 and Trp230 were site-mutated respectively by using over-lap extension.The expression plasmid pET-22b（＋）-PLK was constructed and transformed to Escherichia coli Rosetta for induction and expression,and then the function of the recombinant protein was analyzed after expression product was purified using affinity chromatography.[Results] Compared with the wild type PLK,the PLK activities of the mutantions Thr47,Ile54 and Arg88 were reduced by 82%,58% and 85%,respectively,while the PLK activity of the mutantion Asn121 was hardly affected and the PLK activity of the mutantion Trp230 vanished.[Conclusion] In this study,the significance of selected amino acid residues of side chains on the catalytic function of PLK of B.mori was clarified.