Involvement of a Natural Fusion of a Cytochrome P450 and a Hydrolase in Mycophenolic Acid Biosynthesis

Publication: Research - peer-reviewJournal article – Annual report year: 2012

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@article{aff3a15bcbb1454b8e19f05f6b205515,
title = "Involvement of a Natural Fusion of a Cytochrome P450 and a Hydrolase in Mycophenolic Acid Biosynthesis",
publisher = "American Society for Microbiology",
author = "Hansen, {Bjarne Gram} and Ewelina Mnich and Nielsen, {Kristian Fog} and Nielsen, {Jakob Blæsbjerg} and Nielsen, {Morten Thrane} and Mortensen, {Uffe Hasbro} and Larsen, {Thomas Ostenfeld} and Patil, {Kiran Raosaheb}",
year = "2012",
doi = "10.1128/AEM.07955-11",
volume = "78",
number = "14",
pages = "4908--4913",
journal = "Applied and Environmental Microbiology",
issn = "0099-2240",

}

RIS

TY - JOUR

T1 - Involvement of a Natural Fusion of a Cytochrome P450 and a Hydrolase in Mycophenolic Acid Biosynthesis

A1 - Hansen,Bjarne Gram

A1 - Mnich,Ewelina

A1 - Nielsen,Kristian Fog

A1 - Nielsen,Jakob Blæsbjerg

A1 - Nielsen,Morten Thrane

A1 - Mortensen,Uffe Hasbro

A1 - Larsen,Thomas Ostenfeld

A1 - Patil,Kiran Raosaheb

AU - Hansen,Bjarne Gram

AU - Mnich,Ewelina

AU - Nielsen,Kristian Fog

AU - Nielsen,Jakob Blæsbjerg

AU - Nielsen,Morten Thrane

AU - Mortensen,Uffe Hasbro

AU - Larsen,Thomas Ostenfeld

AU - Patil,Kiran Raosaheb

PB - American Society for Microbiology

PY - 2012

Y1 - 2012

N2 - 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.

AB - 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.

U2 - 10.1128/AEM.07955-11

DO - 10.1128/AEM.07955-11

JO - Applied and Environmental Microbiology

JF - Applied and Environmental Microbiology

SN - 0099-2240

IS - 14

VL - 78

SP - 4908

EP - 4913

ER -