Cloning of the Pactamycin Biosynthetic gene cluster and characterization of a crucial glycosyltransferase prior to a unique cyclopentane ring formation
Cloning of the Pactamycin Biosynthetic gene cluster and characterization of a crucial glycosyltransferase prior to a unique cyclopentane ring formation
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DOI：
10.1038/ja.2007.63
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发表时间：
2007-08-01
影响因子：
3.3
通讯作者：
Eguchi, Tadashi
中科院分区：
文献类型：
--
作者：
Kudo, Furnitaka;Kasama, Yuko;Eguchi, Tadashi
以S-腺苷甲硫氨酸甲基转移酶基因为探针，鉴定了抗肿瘤抗生素帕塔霉素的生物合成基因簇。pct基因簇定位于来自Streptomyces pactum NBRC 13433的34 kb连续DNA，并包含24个开放阅读框。基于生物信息学分析，提出了一条由独特的环戊烷环、3-氨基苯乙酮和6-水杨酸甲酯组成的可能的生物合成途径。推测编码糖基转移酶的pctL基因在独特的环戊烷环形成之前参与3-氨基苯乙酮和UDP-N-乙酰基-α-D-葡糖胺之间的N-糖苷形成。然后在大肠杆菌中异源表达pctL基因，并研究重组PctL蛋白的酶活性。因此，发现PctL蛋白催化形成β-N-糖苷的预期反应。PUL蛋白的酶活性清楚地证实了本发明鉴定的基因簇用于生物合成帕塔霉素。此外，在环戊烷环形成之前的糖基化被认为是结构相关的含环戊烷化合物的生物合成中的一般策略。
The biosynthetic gene (pct) cluster for an antitumor antibiotic pactamycin was identified by use of a gene for putative radical S-adenosylmethionine methyltransferase as a probe. The pct gene cluster is localized to a 34 kb contiguous DNA from Streptomyces pactum NBRC 13433 and contains 24 open reading frames. Based on the bioinformatic analysis, a plausible biosynthetic pathway for pactamycin comprising of a unique cyclopentane ring, 3-aminoacetophenone, and 6-methylsalicylate was proposed. The pctL gene encoding a glycosyltransferase was speculated to be involved in an N-glycoside formation between 3-aminoacetophenone and UDP-N-acetyl-alpha-D-glucosamine prior to a unique cyclopentane ring formation. The pctL gene was then heterologously expressed in Escherichia coli and the enzymatic activity of the recombinant PctL protein was investigated. Consequently, the PctL protein was found to catalyze the expected reaction forming beta-N-glycoside. The enzymatic activity of the PUL protein clearly confirmed that the present identified gene cluster is for the biosynthesis of pactamycin. Also, a glycosylation prior to cyclopentane ring formation was proposed to be a general strategy in the biosynthesis of the structurally related cyclopentane containing compounds.