Acetylaszonalenin Biosynthesis in Neosartorya fischeri IDENTIFICATION OF THE BIOSYNTHETIC GENE CLUSTER BY GENOMIC MINING AND FUNCTIONAL PROOF OF THE GENES BY BIOCHEMICAL INVESTIGATION
Acetylaszonalenin Biosynthesis in Neosartorya fischeri IDENTIFICATION OF THE BIOSYNTHETIC GENE CLUSTER BY GENOMIC MINING AND FUNCTIONAL PROOF OF THE GENES BY BIOCHEMICAL INVESTIGATION
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DOI：
10.1074/jbc.m807606200
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发表时间：
2009-01-02
影响因子：
4.8
通讯作者：
Li, Shu-Ming
中科院分区：
文献类型：
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作者：
Yin, Wen-Bing;Grundmann, Alexander;Li, Shu-Ming
根据从该真菌中分离到的乙酰氨基丁烯的结构信息，通过基因组挖掘，在该真菌的基因组序列中鉴定了一个可能的生物合成基因簇。该簇由编码非核糖体多肽合成酶(AnaPS)、异丙烯基转移酶(AnaPT)和乙酰基转移酶(AnaAT)的三个基因组成。将anaPT和anaAT的编码序列分别克隆到pQE70和pQE60中，并在大肠杆菌中高效表达。将可溶性His(6)融合蛋白纯化到接近均一的水平，并对其进行生化鉴定。酶产物的结构经核磁共振和质谱分析确证。在二甲基烯丙基二磷酸酯存在下，AnaPT催化吲哚部分C3位的(R)-苯并二氮二酮发生反向预烯基化反应，生成阿佐那林。在乙酰辅酶A存在下，AnaAT催化吲哚基团N1位的阿佐那林乙酰化，生成乙酰阿佐那利宁。测定了二甲基烯丙基二磷酸在156mM和(R)-苯二氮杂二酮在232mU时的K-m值，乙酰辅酶A在96mM和Aszonalenin在61mM时的K-m值，AnaPT和AnaAT反应的周转数分别在1.5mU和0.14M·S(-1)。
Based on the structural information of acetylaszonalenin isolated from Neosartorya fischeri, a putative biosynthetic gene cluster was identified in the genome sequence of this fungus by genomic mining. This cluster consists of three genes coding for a putative non-ribosomal peptide synthetase (AnaPS), a prenyltransferase (AnaPT), and an acetyltransferase (AnaAT). The coding sequences of anaPT and anaAT were cloned in pQE70 and pQE60, respectively, and overexpressed in Escherichia coli. The soluble His(6) fusion proteins were purified to near homogeneity and characterized biochemically. The structures of the enzymatic products were elucidated by NMR and mass spectroscopy analysis. AnaPT was found to catalyze the reverse prenylation of (R)-benzodiazepinedione at position C3 of the indole moiety in the presence of dimethylallyl diphosphate, resulting in formation of aszonalenin. AnaAT was found to catalyze the acetylation of aszonalenin at position N1 of the indoline moiety in the presence of acetyl coenzyme A, resulting in formation of acetylaszonalenin. K-m values of AnaPT were determined for dimethylallyl diphosphate at 156 mu M and for (R)-benzodiazepinedione at 232 mu M. K-m values of AnaAT were determined for acetyl coenzyme A at 96 mu M and for aszonalenin at 61 mu M. The turnover numbers of the AnaPT and AnaAT reactions were determined at 1.5 and 0.14 s(-1), respectively.