Nishizawa T, Asayama M, Fujii K, Harada K, Shirai M
Division of Biotechnology, School of Agriculture, Ibaraki University, Ami, Ibaraki, 300-0393, Japan.
J Biochem. 1999 Sep;126(3):520-9. doi: 10.1093/oxfordjournals.jbchem.a022481.
Peptide-synthetase-encoding DNA fragments were isolated by a PCR-based approach from the chromosome of Microcystis aeruginosa K-139, which produces cyclic heptapeptides, 7-desmethylmicrocystin-LR and 3,7-didesmethylmicrocystin-LR. Three open reading frames (mcyA, mcyB, mcyC) encoding microcystin synthetases were identified in the gene cluster. Sequence analysis indicated that McyA (315 kDa) consists of two modules with an N-methylation domain attached to the first and an epimerization domain attached to the second; McyB (242 kDa) has two modules, and McyC (147 kDa) contains one module with a putative C-terminal thioesterase domain. Conserved amino acid sequence motifs for ATP binding, ATP hydrolysis, adenylate formation, and 4'-phosphopantetheine attachment were identified by sequence comparison with authentic peptide synthetase. Insertion mutations in mcyA, generated by homologous recombination, abolished the production of both microcystins in M. aeruginosa K-139. Primer extension analysis demonstrated light-dependent mcy expression. Southern hybridization and partial DNA sequencing analyses of six microcystin-producing and two non-producing Microcystis strains suggested that the microcystin-producing strains contain the mcy gene and the non-producing strains can be divided into two groups, those possessing no mcy genes and those with mcy genes.
采用基于聚合酶链反应（PCR）的方法，从能产生环状七肽、7-去甲基微囊藻毒素-LR和3,7-二去甲基微囊藻毒素-LR的铜绿微囊藻K-139染色体中分离出编码肽合成酶的DNA片段。在该基因簇中鉴定出三个编码微囊藻毒素合成酶的开放阅读框（mcyA、mcyB、mcyC）。序列分析表明，McyA（315 kDa）由两个模块组成，第一个模块连接一个N-甲基化结构域，第二个模块连接一个差向异构化结构域；McyB（242 kDa）有两个模块，McyC（147 kDa）包含一个模块，带有一个推定的C末端硫酯酶结构域。通过与真实肽合成酶的序列比较，鉴定出了ATP结合、ATP水解、腺苷酸形成以及4'-磷酸泛酰巯基乙胺附着的保守氨基酸序列基序。通过同源重组在mcyA中产生的插入突变，消除了铜绿微囊藻K-139中两种微囊藻毒素的产生。引物延伸分析表明mcy的表达依赖于光。对六个产微囊藻毒素的微囊藻菌株和两个不产微囊藻毒素的微囊藻菌株进行Southern杂交和部分DNA测序分析表明，产微囊藻毒素的菌株含有mcy基因，不产微囊藻毒素的菌株可分为两组，一组不含有mcy基因，另一组含有mcy基因。
