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.