Structural and Mechanistic Characterization of the Flavin-Dependent Monooxygenase and Oxidase Involved in Sorbicillinoid Biosynthesis

ACS Chemical Biology, vol. 20, pp. 646–655

Abstract

High Resolution Image Download MS PowerPoint Slide Sorbicillinoids are yellow secondary metabolites synthesized through an elegant combination of enzymatic and spontaneous biochemical processes. The flavin-dependent monooxygenase SorC and oxidase SorD are crucial in this interplay, enabling the generation of a diverse array of functionally complex sorbicillinoids. By solving the crystal structures of SorC and SorD from Penicillium chrysogenum with sorbicillin bound in the active site, we describe the catalytically active binding conformations, crucial for attaining enantioselective and stereoselective control in these enzymatic reactions. The structure of SorC was resolved with the cofactor FAD in its out state, which allowed us to identify key residues that modulate flavin mobility and other conformational changes. Catalytic residues of SorC were also confirmed by detailed characterization of wild-type and several SorC variants. Meanwhile, using a CRISPR/Cas9-based multicopy-genome integration system, we could heterologously express the flavin-dependent oxidase SorD from P. chrysogenum in Aspergillus niger with high yields and purity. This allowed us to obtain the crystal structure of SorD with sorbicillin bound in a viable catalytic conformation. Structural analysis of the obtained complex provided insights into the substrate binding pose and highlighted potentially critical active site residues. Ultimately, having both SorC and SorD at our disposal enabled us to investigate their functions and interplays in the biosynthesis of a vast array of functionally complex sorbicillinoids.

Authors 9

1. Gwen Tjallinks

   University of Groningen · University of Pavia

   Affiliation as printed

   Biomolecular Sciences and Biotechnology Institute, University of Groningen, Nijenborgh 3, Groningen 9747 AG, The Netherlands

   Department of Biology and Biotechnology, University of Pavia, Via Adolfo Ferrata 9, Pavia 27100, Italy

2. Nicolò Angeleri

   University of Pavia

   Affiliation as printed

   Department of Biology and Biotechnology, University of Pavia, Via Adolfo Ferrata 9, Pavia 27100, Italy

3. Quoc‐Thai Nguyen

   University of Medicine and Pharmacy at Ho Chi Minh City

   Affiliation as printed

   Faculty of Pharmacy, University of Medicine and Pharmacy at Ho Chi Minh City, 41 Dinh Tien Hoang Street, Ben Nghe Ward, District 1, Ho Chi Minh City 70000, Vietnam

4. Barbara Mannucci

   University of Pavia

   Affiliation as printed

   Department of Biology and Biotechnology, University of Pavia, Via Adolfo Ferrata 9, Pavia 27100, Italy

5. Mark Arentshorst Aachen Fungal Genetics and Biotechnology Institute of Biology Leiden

   Leiden University

   Affiliation as printed

   Fungal Genetics and Biotechnology, Institute of Biology Leiden, Leiden University, Sylviusweg 72, Leiden 2333 BE, The Netherlands

6. Jaap Visser Aachen Fungal Genetics and Biotechnology Institute of Biology Leiden

   Leiden University

   Affiliation as printed

   Fungal Genetics and Biotechnology, Institute of Biology Leiden, Leiden University, Sylviusweg 72, Leiden 2333 BE, The Netherlands

7. Arthur F. J. Ram Aachen Fungal Genetics and Biotechnology Institute of Biology Leiden

   Leiden University

   Affiliation as printed

   Fungal Genetics and Biotechnology, Institute of Biology Leiden, Leiden University, Sylviusweg 72, Leiden 2333 BE, The Netherlands

8. Marco W. Fraaije corresponding

   University of Groningen

   Affiliation as printed

   Biomolecular Sciences and Biotechnology Institute, University of Groningen, Nijenborgh 3, Groningen 9747 AG, The Netherlands

9. Andrea Mattevi corresponding

   University of Pavia

   Affiliation as printed

   Department of Biology and Biotechnology, University of Pavia, Via Adolfo Ferrata 9, Pavia 27100, Italy