Identification of the Verruculogen Prenyltransferase FtmPT3 by a Combination of Chemical, Bioinformatic and Biochemical Approaches
Identification of the Verruculogen Prenyltransferase FtmPT3 by a Combination of Chemical, Bioinformatic and Biochemical Approaches
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DOI：
10.1002/cbic.201200523
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发表时间：
2012-11-26
期刊：
影响因子：
3.2
通讯作者：
Li, Shu-Ming
中科院分区：
文献类型：
--
作者：
Mundt, Kathrin;Wollinsky, Beate;Li, Shu-Ming
以往的研究表明，verruculogen是烟曲霉（Aspergillus fumigatus）和费氏奈氏酵母（Neapritorya fischeri）中烟曲霉素型生物碱生物合成基因簇的终产物。本研究从N. fischeri。这导致了责任基因的鉴定，ftmPT 3，在verruculogen脂肪族羟基的O-异戊二烯化。该基因位于N. fischeri比确定的集群。通过融合PCR扩增出ftmPT 3的编码序列，并在大肠杆菌中高效表达。可溶性His 8-FtmPT 3与疣状蛋白和二甲基烯丙基二磷酸（DMAPP）的酶产物通过NMR和MS分析明确地鉴定为烟曲霉素A。分别在5.7和61.5 μ M下测定了Verruculogen和DMAPP的FtmPT 3的KM值。根据Verruculogen和DMAPP的动力学参数计算平均转换数（kcat）为0.069 s-1。FtmPT 3还接受烟曲霉素A的生物合成前体，例如烟曲霉素B和12，13-二羟基烟曲霉素C作为底物，并催化它们的异戊烯化。
Previous studies showed that verruculogen is the end product of a biosynthetic gene cluster for fumitremorgin-type alkaloids in Aspergillus fumigatus and Neosartorya fischeri. In this study, we isolated fumitremorgin A from N. fischeri. This led to the identification of the responsible gene, ftmPT3, for O-prenylation of an aliphatic hydroxy group in verruculogen. This gene was found at a different location in the genome of N. fischeri than the identified cluster. The coding sequence of ftmPT3 was amplified by fusion PCR and overexpressed in Escherichia coli. The enzyme product of the soluble His8-FtmPT3 with verruculogen and dimethylallyl diphosphate (DMAPP) was identified unequivocally as fumitremorgin A by NMR and MS analyses. KM values of FtmPT3 were determined for verruculogen and DMAPP at 5.7 and 61.5 mu M, respectively. Average turnover number (kcat) was calculated from kinetic parameters of verruculogen and DMAPP to be 0.069 s-1. FtmPT3 also accepted biosynthetic precursors of fumitremorgin A, for example, fumitremorgin B and 12,13-dihydroxyfumitremorgin C, as substrates and catalyses their prenylation.