Pentalenolactone biosynthesis.: Molecular cloning and assignment of biochemical function to PtlH, a non-heme iron dioxygenase of Streptomyces avermitilis
Pentalenolactone biosynthesis.: Molecular cloning and assignment of biochemical function to PtlH, a non-heme iron dioxygenase of Streptomyces avermitilis
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DOI：
10.1021/ja061469i
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发表时间：
2006-05-24
影响因子：
15
通讯作者：
Cane, David E.
中科院分区：
文献类型：
--
作者：
You, Zheng;Omura, Satoshi;Cane, David E.
利用PCR方法克隆了阿维链霉菌五烯内酯基因簇中ptlh （SAV2991）基因编码的羟化酶，并在大肠杆菌中以n端his6标签蛋白表达。重组PtlH与（±）-1-脱氧戊烯酸(5)在Fe（II）、α-酮戊二酸和o2(−)-11β-羟基-1-脱氧戊烯酸(8)存在下孵育，其结构和立体化学通过1h、13C、COSY、HMQC、HMBC和NOESY NMR组合测定。稳态动力学参数ekcat= 4.2±0.6 s-1, km(5) = 0.57±0.19 mm。8是倍半萜类五烯烯(3)转化为五烯内酯(1)的新中间体。
The hydroxylase encoded by theptlH(SAV2991) gene from the pentalenolactone gene cluster ofStreptomyces avermitiliswas cloned by PCR and expressed inEscherichia colias an N-terminal His6-tag protein. Incubation of recombinant PtlH with (±)-1-deoxypentalenic acid (5) in the presence of Fe(II), α-ketoglutarate, and O2gave (−)-11β-hydroxy-1-deoxypentalenic acid (8), whose structure and stereochemistry were determined by a combination of1H,13C, COSY, HMQC, HMBC, and NOESY NMR. The steady-state kinetic parameters werekcat= 4.2 ± 0.6 s-1andKm(5) = 0.57 ± 0.19 mM.8is a new intermediate in the conversion of the sesquiterpene pentalenene (3) to pentalenolactone (1).