Heterologous Expression and Engineering of the Nitrogenase Cofactor Biosynthesis Scaffold NifEN. (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. Volume 132:Number 17(2020)
• Page Start: 6954
• Page End: 6960
• Publication Date: 2020-03-03
• Subjects: Bioanorganische Chemie -- Nitrogenase -- Metalloenzyme -- Metalloproteine -- Protein-Engineering; Chemistry -- Periodicals; 540
• DOI: 10.1002/ange.201916598
• Languages: English
• ISSNs: 0044-8249

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 ähnelt in Struktur und Funktion der katalytischen Komponente von Nitrogenase, NifDK. Die Expression und Entwicklung einer NifEN‐Variante, die NifDK imitiert, indem der Cofaktor an einer analogen Bindungsstelle erhalten bleibt, wird für E. coli vorgestellt. Die Beobachtung demonstriert die Realisierbarkeit der Erzeugung eines funktionellen NifDK‐Äquivalents auf einem NifEN‐Templat.