Center for Microbial Biotechnology, Department of Systems Biology, Technical University of Denmark, Kgs. Lyngby, Denmark.
Appl Environ Microbiol. 2012 Jul;78(14):4908-13. doi: 10.1128/AEM.07955-11. Epub 2012 Apr 27.
Mycophenolic acid (MPA) is a fungal secondary metabolite and the active component in several immunosuppressive pharmaceuticals. The gene cluster coding for the MPA biosynthetic pathway has recently been discovered in Penicillium brevicompactum, demonstrating that the first step is catalyzed by MpaC, a polyketide synthase producing 5-methylorsellinic acid (5-MOA). However, the biochemical role of the enzymes encoded by the remaining genes in the MPA gene cluster is still unknown. Based on bioinformatic analysis of the MPA gene cluster, we hypothesized that the step following 5-MOA production in the pathway is carried out by a natural fusion enzyme MpaDE, consisting of a cytochrome P450 (MpaD) in the N-terminal region and a hydrolase (MpaE) in the C-terminal region. We verified that the fusion gene is indeed expressed in P. brevicompactum by obtaining full-length sequence of the mpaDE cDNA prepared from the extracted RNA. Heterologous coexpression of mpaC and the fusion gene mpaDE in the MPA-nonproducer Aspergillus nidulans resulted in the production of 5,7-dihydroxy-4-methylphthalide (DHMP), the second intermediate in MPA biosynthesis. Analysis of the strain coexpressing mpaC and the mpaD part of mpaDE shows that the P450 catalyzes hydroxylation of 5-MOA to 4,6-dihydroxy-2-(hydroxymethyl)-3-methylbenzoic acid (DHMB). DHMB is then converted to DHMP, and our results suggest that the hydrolase domain aids this second step by acting as a lactone synthase that catalyzes the ring closure. Overall, the chimeric enzyme MpaDE provides insight into the genetic organization of the MPA biosynthesis pathway.
麦考酚酸（MPA）是一种真菌次级代谢产物，也是几种免疫抑制药物的活性成分。最近在短密青霉中发现了编码 MPA 生物合成途径的基因簇，表明第一步由多酮合酶 MpaC 催化，产生 5-甲基奥尔酸（5-MOA）。然而，该 MPA 基因簇中其余基因编码的酶的生化作用仍然未知。基于对 MPA 基因簇的生物信息学分析，我们假设该途径中 5-MOA 生成后的步骤是由天然融合酶 MpaDE 完成的，该酶由 N 端的细胞色素 P450（MpaD）和 C 端的水解酶（MpaE）组成。我们通过从提取的 RNA 中获得全长 mpaDE cDNA 来证实融合基因确实在短密青霉中表达。在非 MPA 产生菌构巢曲霉中异源共表达 mpaC 和融合基因 mpaDE 导致 5,7-二羟基-4-甲基邻苯二甲酸（DHMP）的生成，这是 MPA 生物合成的第二个中间产物。对共表达 mpaC 和 mpaDE 的 mpaD 部分的菌株进行分析表明，该 P450 催化 5-MOA 羟化为 4,6-二羟基-2-（羟甲基）-3-甲基苯甲酸（DHMB）。然后 DHMB 转化为 DHMP，我们的结果表明，水解酶结构域通过充当催化环闭合的内酯合酶来辅助这第二步。总体而言，嵌合酶 MpaDE 为 MPA 生物合成途径的遗传组织提供了深入了解。
