Zentrum für Synthetische Mikrobiologie, Philipps-Universität Marburg, 35032 Marburg, Germany.
Institut für Pharmazeutische Biologie und Biotechnologie, Philipps-Universität Marburg, Deutschhausstrasse 17A, 35037 Marburg, Germany.
Microbiology (Reading). 2013 Oct;159(Pt 10):2169-2179. doi: 10.1099/mic.0.069542-0. Epub 2013 Jul 11.
A putative prenyltransferase gene, NFIA_043650, was amplified from Neosartorya fischeri NRRL 181 and cloned into the expression vector pQE60. The deduced polypeptide consisting of 445 amino acids with a molecular mass of 51 kDa was overproduced in Escherichia coli and purified as His6-tagged protein to near homogeneity. The purified soluble protein was subsequently assayed with potential aromatic substrates in the presence of dimethylallyl diphosphate. HPLC analysis of the reaction mixtures revealed acceptance of all tested tryptophan-containing cyclic dipeptides. Isolation and structural elucidation of enzyme products of five selected substrates indicated a reverse C2-prenylation on the indole nucleus, proving the enzyme to be a cyclic dipeptide C2-prenyltransferase (CdpC2PT). Differing significantly from two known brevianamide F reverse C2-prenyltransferases NotF and BrePT which use cyclo-l-Trp-l-Pro as their preferred substrate, CdpC2PT showed a clear substrate preference for (S)-benzodiazepinedinone and cyclo-l-Trp-l-Trp with KM values of 84.1 and 165.2 µM and turnover numbers at 0.63 and 0.30 s(-1), respectively. A possible role of CdpC2PT in the biosynthesis of fellutanines is discussed.
从 Neosartorya fischeri NRRL 181 中扩增出一个假定的prenyltransferase 基因 NFIA_043650，并将其克隆到表达载体 pQE60 中。该推定的多肽由 445 个氨基酸组成，分子量为 51 kDa，在大肠杆菌中大量表达，并作为 His6 标记蛋白纯化至近乎均一。随后，用潜在的芳香族底物和二甲基烯丙基二磷酸在纯化的可溶性蛋白中进行测定。反应混合物的 HPLC 分析表明，所有测试的含色氨酸的环状二肽都被接受。对五个选定底物的酶产物进行分离和结构阐明表明，在吲哚核上发生了反向 C2-prenylation，证明该酶是一种环状二肽 C2-prenyltransferase（CdpC2PT）。与两种已知的 brevianamide F 反向 C2-prenyltransferases NotF 和 BrePT 显著不同，它们使用环-l-Trp-l-Pro 作为其首选底物，CdpC2PT 对 (S)-benzodiazepinedinone 和环-l-Trp-l-Trp 表现出明显的底物偏好，KM 值分别为 84.1 和 165.2 μM，周转率分别为 0.63 和 0.30 s(-1)。讨论了 CdpC2PT 在 fellutanines 生物合成中的可能作用。
