title: Involvement of a natural fusion of a cytochrome P450 and a hydrolase in mycophenolic acid biosynthesis.
abstract: 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.
authors.affiliation: Center for Microbial Biotechnology, Department of Systems Biology, Technical University of Denmark, Kgs. Lyngby, Denmark.
authors.forename: Bjarne Gram
authors.initials: BG
authors.lastname: Hansen
authors.forename: Ewelina
authors.initials: E
authors.lastname: Mnich
authors.forename: Kristian Fog
authors.initials: KF
authors.lastname: Nielsen
authors.forename: Jakob Blæsbjerg
authors.initials: JB
authors.lastname: Nielsen
authors.forename: Morten Thrane
authors.initials: MT
authors.lastname: Nielsen
authors.forename: Uffe Hasbro
authors.initials: UH
authors.lastname: Mortensen
authors.forename: Thomas Ostenfeld
authors.initials: TO
authors.lastname: Larsen
authors.forename: Kiran Raosaheb
authors.initials: KR
authors.lastname: Patil
mesh_terms: D000595:Amino Acid Sequence; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism; D006867:Hydrolases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D009173:Mycophenolic Acid / Q000096:biosynthesis*; D010407:Penicillium / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D016415:Sequence Alignment
chemical_list: D005656:Fungal Proteins; D011993:Recombinant Fusion Proteins; D003577:Cytochrome P-450 Enzyme System; D006867:Hydrolases; D009173:Mycophenolic Acid