Heterologous Expression and Engineering of the Nitrogenase Cofactor Biosynthesis Scaffold NifEN

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.

Automated Laboratory Growth Assessment and Maintenance of Azotobacter vinelandii

“…These include the study of aerobic respiration (Poole & Hill, 1997), hydrogen uptake and production (Kow & Burris, 1984;Noar, Loveless, Navarro-Herrero, Olson, & Bruno-Barcena, 2015), polymer production (Clementi, 1997;Galindo, Pena, Nunez, Segura, & Espin, 2007), and, importantly, the genetics and biochemistry of nitrogenasecatalyzed nitrogen fixation (Dos Santos, 2019;Hoffman et al, 2014;Noar & Bruno-Barcena, 2018). Due to the challenge of heterologous expression of functional nitrogenases A. vinelandii has been used for their expression, purification, and biochemical characterization (Hoffman et al, 2014;Lee, Ribbe, & Hu, 2019;Solomon et al, 2020). Furthermore, A. vinelandii, which exhibits diauxic metabolism (George, Costenbader, & Melton, 1985) and grows in a wide range of oxygen levels (Dingler, Kuhla, Wassink, & Oelze, 1988), is an important model for studying nitrogen fixation in diverse environments.…”

Reconstruction of Nitrogenase Predecessors Suggests Origin from Maturase-Like Proteins

“…All are in or proximal to the nitrogenase active site or M-cluster insertion funnel. Some have specific inferred or experimentally determined functional roles in nitrogenases, including as an M-cluster ligand (site 442; Kim and Rees 1992 ), a “lock” to hold the M-cluster within the active site (site 444; Hu et al 2008 ; Solomon et al 2020 ), and a “lid” at the cluster insertion funnel opening (site 362; Hu et al 2008 ). Finally, our set of divergent sites includes the maturase cluster precursor ligand, site 48 ( Kaiser et al 2011 ).…”

“…These site-wise D-scores were assessed specifically along the phylogenetic transect between the last common ancestor of nitrogenases and the last common ancestor of all nitrogenase and maturases ( supplementary table S3, Supplementary Material online). Certain divergent sites that become “nitrogenase-like” early (i.e., prior to the nitrogenase ancestor) include site 195 (important for N 2 substrate binding; Kim et al 1995 ), site 444 (locks the M-cluster in the nitrogenase active site; Hu et al 2008 ; Solomon et al 2020 ), and site 359 (helps form the cluster insertion funnel; Hu et al 2006 ). By contrast, site 48 (involved with L-cluster binding in maturases; Kaiser et al 2011 ), as well as sites 361 and 362 (involved with M-cluster insertion at the nitrogenase active site; Hu et al 2008 ), remain primarily “maturase-like” until the nitrogenase last common ancestor.…”

Reconstruction of nitrogenase predecessors suggests origin from maturase-like proteins

“…These site-wise D-scores were assessed specifically along the phylogenetic transect between the last common ancestor of nitrogenases and the last common ancestor of all nitrogenase and maturases (supplementary table S3). Certain divergent sites that become “nitrogenase-like” early (i.e., prior to the nitrogenase ancestor) include site 195 (important for N 2 substrate binding (Kim et al 1995)), site 444 (locks the M-cluster in the nitrogenase active site (Hu et al 2008; Solomon et al 2020)), and site 359 (helps form the cluster insertion funnel (Hu et al 2006)). By contrast, site 48 (involved with L-cluster binding in maturases (Kaiser et al 2011)), as well as sites 361 and 362 (involved with M-cluster insertion at the nitrogenase active site (Hu et al 2008)), remain primarily “maturase-like” until the nitrogenase last common ancestor.…”