变铅青链霉菌异柠檬酸脱氢酶的异源表达及序列分析

Heterologous Expression and Sequence Analysis of Isocitrate Dehydrogenase from Streptomyces lividans TK54

通过同源性引物成功扩增和克隆了变铅青链霉菌(Streptomyces lividans)TK54的异柠檬酸脱氢酶(isocitratedehydrogenase,IDH)(简称SlIDH)基因icd(GenBank登录号为EU661252)。icd的起始密码子为GTG,GC含量为69.55%,显示了链霉菌基因的高GC含量特征,实现了SlIDH在E.coli中的异源高效表达。0.5 mmol/L的IPTG为最佳诱导条件。SlIDH的分子量约为80 kD。在Mn^(2+)或Mg^(2+)条件下,SlIDH以NADP^+为辅酶时的活性分别为7.94 U/mg及4.00 U/mg,以NAD^+为辅酶时的活性分别为0.58 U/mg及0.27 U/mg,SlIDH更偏爱以NADP^+为辅酶。与不同种属单体IDH的氨基酸序列比对显示,SlIDH与单体IDH的序列一致性均在60%以上。因此本工作首次以实验性证据初步鉴定了SlIDH为NADP-依赖型单体IDH。本工作为进一步探索单体IDH的结构与功能以及单体IDH与同源二聚体IDH的进化关系奠定了基础。

The icd gene （GenBank accession No. EU623595） encoding isocitrate dehydrogenase （IDH） from Streptomyces lividans TK54 （S/IDH） was amplified and cloned using homologous primers. The icd gene start codon is GTG and its GC content is 69.55 % which is consistent with the high-GC characteristics in genes from Streptomyces. SlIDH was overexpressed heterologously in Escherichia coli. The best condition for induction was 0.5 mmol/L IPTG. The molecular weight of SIlDH was about 80 kD. In the presence of Mn^＋2 or Mg^2 ＋ , the S/IDH activity was 7.94 U/mg and 4.00 U/ mg using NADP^＋ as a coenzyme, respectively. When using NAD^＋ as a coenzyme, the SlIDH activity was 0.58 U/mg and 0. 27 U/mg, respectively. It indicated that SlIDH much prefers utilizing NADP^＋ as its cofactor. The sequence identities between SlIDH and monomeric IDH homologs from various species were all above 60 % based on the comparison of their amino acid sequences. Therefore, these results provide the first experimental evidence that SlIDH is a NADP-dependent monomeric IDH. This work will be a fundament for further investigating the relationships between the structure and function of monomeric IDH and the evolutionary relationships between monomeric IDH and homodimeric IDH.