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Heterologous Expression and Engineering of the Nitrogenase Cofactor Biosynthesis Scaffold NifEN
Author(s) -
Solomon Joseph B.,
Lee Chi Chung,
Jasniewski Andrew J.,
Rasekh Mahtab F.,
Ribbe Markus W.,
Hu Yilin
Publication year - 2020
Publication title -
angewandte chemie
Language(s) - English
Resource type - Journals
eISSN - 1521-3757
pISSN - 0044-8249
DOI - 10.1002/ange.201916598
Subject(s) - azotobacter vinelandii , nitrogenase , cofactor , heterologous expression , biochemistry , biosynthesis , heterologous , escherichia coli , enzyme , chemistry , biology , bacteria , gene , nitrogen fixation , genetics , recombinant dna
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.