Quaderer Richard, Omura Satoshi, Ikeda Haruo, Cane David E
Department of Chemistry, Brown University, Box H, Providence, Rhode Island 02912-9108, USA.
J Am Chem Soc. 2006 Oct 11;128(40):13036-7. doi: 10.1021/ja0639214.
A gene cluster encoding all of the enzymes for the biosynthesis of the antibiotic pentalenolactone (1) has recently been identified in Streptomyces avermitilis. The biosynthetic gene cluster contains the ptlI (SAV2999) gene which encodes a cytochrome P450 (CYP183A1). PtlI was cloned by PCR and expressed in Escherichia coli as a C-terminal His6-tag protein. Recombinant PtlI bound pentalenene (3) with high affinity (KD = 1.44 +/- 0.14 muM). Incubation of recombinant PtlI with (+/-)-3 in the presence of NADPH, E. coli flavodoxin and flavodoxin reductase, and O2 resulted in conversion to a single enantiomer of pentalen-13-al (7), by stepwise allylic oxidation via pentalen-13-ol (6). The steady-state kinetic parameters for the oxidation of pentalenene (3) to pentalen-13-ol (6) were kcat = 0.503 +/- 0.006 min-1 and Km = 3.33+/-0.62 muM for 3.
最近在阿维链霉菌中发现了一个编码抗生素戊内酯（1）生物合成所有酶的基因簇。该生物合成基因簇包含ptlI（SAV2999）基因，其编码一种细胞色素P450（CYP183A1）。通过PCR克隆PtlI，并作为C端His6标签蛋白在大肠杆菌中表达。重组PtlI与戊烯（3）具有高亲和力结合（KD = 1.44 +/- 0.14 μM）。在NADPH、大肠杆菌黄素氧还蛋白和黄素氧还蛋白还原酶以及O2存在下，将重组PtlI与（±）-3一起孵育，通过戊烯-13-醇（6）逐步烯丙基氧化，转化为戊烯-13-醛（7）的单一对映体。将戊烯（3）氧化为戊烯-13-醇（6）的稳态动力学参数为：对于3，kcat = 0.503 +/- 0.006 min-1，Km = 3.33 +/- 0.62 μM。
