变铅青链霉菌异柠檬酸脱氢酶的异源表达及序列分析

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(isocitrate dehydrogenase, 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.

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

The icd gene (GenBank accession No. EU623595) encoding isocitrate dehydrogenase (IDH) from Streptomyces lividans TK54 (SlIDH) 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 SlIDH was about 80 kD. In the presence of Mn~(2+) or Mg~(2+), the SlIDH 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.