Department of Chemistry, National Chung Hsing University, Taichung, Taiwan 40227, Rep. of China.
Chemistry. 2012 May 14;18(20):6238-49. doi: 10.1002/chem.201102825. Epub 2012 Mar 30.
Neocarzinostatin is an antibiotic chromoprotein produced by Streptomyces carzinostaticus. Its enediyne-containing chromophore exhibits high DNA cleavage activity and belongs to one of the most potent categories of antitumor agents. The labile chromophore is readily inactivated by environmental thiols including the most abundant glutathione. How the microorganism preserves the secreted antibiotic and at the same time is immune to its toxicity are of interest. Site-directed mutagenesis studies of the neocarzinostatin protein have shown that residues D33 and D99 play primary and secondary roles, respectively, in preserving neocarzinostatin from acidic glutathione whereas D79 and other residues around the opening of the binding cleft have an insignificant effect. Biothiol analyses revealed that cells of S. carzinostaticus produced no glutathione, but instead neutral mycothiol, which is known to serve functions analogous to glutathione. Mycothiol was the only neutral-charged thiol produced by the organism; all other identified biothiols carried at least partial negative charges. When the bacteria were cultured under conditions that stimulated the biosynthesis of neocarzinostatin, the yield of mycothiol increased significantly, which suggests mycothiol-dependent cellular detoxification. Treating neocarzinostatin samples with the cell extract that retained active sulfhydryls led to efficient drug inactivation, which indicates that mycothiol is allowed to approach the protein-bound chromophore. The anionic side-chains of D33 and D99 in the neocarzinostatin protein played two critical roles in a single thiol-screening operation: Preserving the antibiotic for defense and survival by rejecting the ubiquitous glutathione through charge-charge repulsion in the outer-cell environment and detoxifying the toxin in the inner-cell body for self-resistance by accepting the cell-produced neutral mycothiol.
新制癌菌素是一种由链霉菌产生的抗生素色蛋白。其含有烯二炔的发色团表现出很高的 DNA 切割活性，属于最有效的抗肿瘤药物之一。不稳定的发色团很容易被包括最丰富的谷胱甘肽在内的环境硫醇失活。微生物如何保存分泌的抗生素，同时又能免受其毒性的影响，这是一个很有趣的问题。对新制癌菌素蛋白的定点突变研究表明，残基 D33 和 D99 分别在酸性谷胱甘肽中保护新制癌菌素免受失活方面起主要和次要作用，而 D79 和结合裂缝开口周围的其他残基则没有影响。生物硫醇分析表明，链霉菌细胞不产生谷胱甘肽，而是产生中性的麦硫醇，麦硫醇已知具有类似于谷胱甘肽的功能。麦硫醇是该生物产生的唯一带中性电荷的硫醇；所有其他鉴定出的生物硫醇都带有至少部分负电荷。当细菌在刺激新制癌菌素生物合成的条件下培养时，麦硫醇的产量显著增加，这表明麦硫醇依赖的细胞解毒作用。用保留活性巯基的细胞提取物处理新制癌菌素样品会导致药物失活效率很高，这表明麦硫醇可以接近蛋白结合的发色团。新制癌菌素蛋白中的 D33 和 D99 的阴离子侧链在单个巯基筛选操作中发挥了两个关键作用：通过在外细胞环境中通过电荷-电荷排斥来拒绝普遍存在的谷胱甘肽，从而为防御和生存保留抗生素；通过接受细胞产生的中性麦硫醇，在细胞内体中解毒毒素，从而实现自身抵抗。
