Heterologous Expression and Engineering of the Nitrogenase Cofactor Biosynthesis Scaffold NifEN. Issue 17 (3rd March 2020)

• Record Type: Journal Article
• Authors: Solomon, Joseph B.; Lee, Chi Chung; Jasniewski, Andrew J.; Rasekh, Mahtab F.; Ribbe, Markus W.; Hu, Yilin
• Is Part Of: Angewandte Chemie international edition. Volume 59:Issue 17(2020)
• Page Start: 6887
• Page End: 6893
• Publication Date: 2020-03-03
• Subjects: bioinorganic chemistry -- nitrogenase -- metalloenzymes -- metalloproteins -- protein engineering; Chemistry -- Periodicals; 540
• DOI: 10.1002/anie.201916598
• Languages: English
• ISSNs: 1433-7851

Abstract

Abstract: NifEN plays a crucial role in the biosynthesis of nitrogenase, catalyzing the final step of cofactor maturation prior to delivering the cofactor to NifDK, the catalytic component of nitrogenase. The difficulty in expressing NifEN, a complex, heteromultimeric metalloprotein sharing structural/functional homology with NifDK, is a major challenge in the heterologous expression of nitrogenase. Herein, we report the expression and engineering of Azotobacter vinelandii NifEN in Escherichia coli . Biochemical and spectroscopic analyses demonstrate the integrity of the heterologously expressed NifEN in composition and functionality and, additionally, the ability of an engineered NifEN variant to mimic NifDK in retaining the matured cofactor at an analogous cofactor‐binding site. This is an important step toward piecing together a viable pathway for the heterologous expression of nitrogenase and identifying variants for the mechanistic investigation of this enzyme. Abstract : NifEN resembles the catalytic component of nitrogenase, NifDK, in structure and function. The expression and engineering of a NifEN variant, which mimics NifDK in retaining the cofactor at an analogous binding site, in E. coli is reported. This observation demonstrates the feasibility of generating a functional NifDK equivalent on a NifEN template, which could facilitate future heterologous expression and mechanistic investigation of nitrogenase.