Cane David E, Watt Rory M
Department of Chemistry, Brown University, Providence, RI 02912, USA.
Proc Natl Acad Sci U S A. 2003 Feb 18;100(4):1547-51. doi: 10.1073/pnas.0337625100. Epub 2003 Jan 29.
The PCR has been used to amplify a 2,181-bp ORF from Streptomyces coelicolor A3(2), designated SC9B1.20 (= SCO6073), encoding a protein of 726 amino acids and showing significant sequence similarity at the deduced amino acid level in both the N-terminal and C-terminal halves to the known sesquiterpene synthase pentalenene synthase. The full-length recombinant protein was expressed at high levels in Escherichia coli and shown to catalyze the Mg(2+)-dependent conversion of farnesyl diphosphate to the sesquiterpene alcohol (4S, 7R)-germacra-1 (10)E, 5E-diene-11-ol. The enzymatic cyclization had a k(cat) of 6.2 +/- 0.5 x 10(-3) s(-1) and a K(m) for farnesyl diphosphate of 62 +/- 8 nM. Expression of the N-terminal (366 amino acids) domain of the SC9B1.20 protein also gave a fully functional cyclase which converted farnesyl diphosphate to the identical sesquiterpene alcohol with a slightly lower k(cat) of 3.2 +/- 0.4 x 10(-3) s(-1) and a twofold greater k(m) of 115 +/- 14 nM. By contrast, the expressed C-terminal domain of SC9B1.20 had no farnesyl diphosphate cyclase activity. The formation of the germacradienol seems to be the committed step in the formation of geosmin, the characteristic odoriferous constituent of Streptomyces species.
聚合酶链式反应（PCR）已用于从天蓝色链霉菌A3(2)中扩增出一个2181 bp的开放阅读框（ORF），命名为SC9B1.20（= SCO6073），其编码一个726个氨基酸的蛋白质，并且在推导的氨基酸水平上，该蛋白质的N端和C端两半部分与已知的倍半萜合酶古巴烯合酶均显示出显著的序列相似性。全长重组蛋白在大肠杆菌中高水平表达，并显示出催化法呢基二磷酸（FPP）依赖镁离子的转化，生成倍半萜醇（4S, 7R）-吉马-1(10)E, 5E-二烯-11-醇。酶促环化反应的催化常数（k(cat)）为6.2±0.5×10⁻³ s⁻¹，FPP的米氏常数（K(m)）为62±8 nM。SC9B1.20蛋白的N端（366个氨基酸）结构域的表达也产生了一种功能完全的环化酶，该酶将FPP转化为相同的倍半萜醇，其k(cat)略低，为3.2±0.4×10⁻³ s⁻¹，K(m)则是其两倍，为115±14 nM。相比之下，SC9B1.20蛋白表达的C端结构域没有FPP环化酶活性。吉马二烯醇的形成似乎是土腥味素形成过程中的关键步骤，土腥味素是链霉菌属物种特有的有气味成分。
