PENTALENENE SYNTHASE - PURIFICATION, MOLECULAR-CLONING, SEQUENCING, AND HIGH-LEVEL EXPRESSION IN ESCHERICHIA-COLI OF A TERPENOID CYCLASE FROM STREPTOMYCES UC5319
PENTALENENE SYNTHASE - PURIFICATION, MOLECULAR-CLONING, SEQUENCING, AND HIGH-LEVEL EXPRESSION IN ESCHERICHIA-COLI OF A TERPENOID CYCLASE FROM STREPTOMYCES UC5319
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DOI：
10.1021/bi00185a024
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发表时间：
1994-05-17
期刊：
影响因子：
2.9
通讯作者：
HUBBARD, BR
中科院分区：
文献类型：
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作者：
CANE, DE;SOHNG, JK;HUBBARD, BR
从链霉菌UC 5319中纯化并环戊二烯合酶，其催化法呢基二磷酸（1）环化为三环倍半萜烃并环戊二烯（2）。一个450 bp的杂交探针，通过PCR扩增的基因组DNA，使用引物的基础上的N-末端和内部胰蛋白酶肽序列数据的并环戊二烯合酶，被用来筛选链霉菌基因组DNA的质粒和噬菌体DNA文库，导致在分离和测序的完整的并环戊二烯合酶基因。利用PCR将并环戊二烯合酶基因插入到T7表达载体pLM 1中。在表达宿主大肠杆菌BL 21（DE 3）中克隆所得构建体得到表达大于10%可溶性蛋白的并环戊二烯合酶的转化体。重组酶已被纯化，并进行了初步的物理和动力学表征。该重组酶在各方面似乎与天然链霉菌合酶相同，并显示下列稳态动力学参数：K-m = 0.31 +/-0.05 μ M，k（cat）= 0.32 +/-0.02 s（-1），K-I（PPi）= 3.2 +/-0.6 μ M。这两种酶都具有用于催化的Mg 2+的绝对需求和8.2-8.4的最适pH。如通过SDS-PAGE测定的，两种蛋白质具有41-42 kDa的M（r）值。
Pentalenene synthase, which catalyzes the cyclization of farnesyl diphosphate (1) to the tricyclic sesquiterpene hydrocarbon pentalenene (2), was purified from Streptomyces UC5319. A 450-bp hybridization probe, generated by PCR amplification of genomic DNA using primers based on N-terminal and internal tryptic peptide sequence data for pentalenene synthase, was used to screen both plasmid and phage DNA libraries of Streptomyces genomic DNA, resulting in the isolation and sequencing of the complete pentalenene synthase gene. PCR was used to insert the pentalenene synthase gene into the T7 expression vector pLM1. Cloning of the resulting construct in the expression host Escherichia coli BL21(DE3) gave transformants that expressed pentalenene synthase as greater than 10% of soluble protein. The recombinant enzyme has been purified, and initial physical and kinetic characterization has been performed. The recombinant enzyme appears to be identical in every respect with the native Streptomyces synthase and exhibits the following steady-state kinetic parameters: K-m = 0.31 +/- 0.05 mu M, k(cat) = 0.32 +/- 0.02 s(-1), K-I(PPi) = 3.2 +/- 0.6 mu M. Both enzymes have an absolute requirement of Mg2+ for catalysis and an optimum pH of 8.2-8.4. Both proteins have M(r) values of 41-42 kDa, as determined by SDS-PAGE.