Abstract
One of the main hurdles to engineer nitrogenase in a non-diazotrophic host is achieving NifB activity. NifB is an extremely unstable and oxygen sensitive protein that catalyzes a low-potential SAM-radical dependent reaction. The product of NifB activity is called NifB-co, a complex [8Fe-9S-C] cluster that serves as obligate intermediate in the biosyntheses of the active-site cofactors of all known nitrogenases. Here we study the diversity and phylogeny of naturally occurring NifB proteins, their protein architecture and the functions of the distinct NifB domains in order to understand what defines a catalytically active NifB. Focus is on NifB from the thermophile Chlorobium tepidum (two-domain architecture), the hyperthermophile Methanocaldococcus infernus (single-domain architecture) and the mesophile Klebsiella oxytoca (two-domain architecture), showing in silico characterization of their nitrogen fixation (nif) gene clusters, conserved NifB motifs, and functionality. C. tepidum and M. infernus NifB were able to complement an Azotobacter vinelandii (ΔnifB) mutant restoring the Nif+ phenotype and thus demonstrating their functionality in vivo. In addition, purified C. tepidum NifB exhibited activity in the in vitro NifB-dependent nitrogenase reconstitution assay. Intriguingly, changing the two-domain K. oxytoca NifB to single-domain by removal of the C-terminal NifX-like extension resulted in higher in vivo nitrogenase activity, demonstrating that this domain is not required for nitrogen fixation in mesophiles.
Highlights
Nitrogen is abundant on Earth, most of it is in the form of dinitrogen (N≡N or N2)
The 390 putative NifB sequences found in the Structure and Function Linkage Database (SFLD) (Akiva et al, 2014) are shown in the Supplementary Table 1
By aligning experimentally proven NifB proteins from A. vinelandii (NifBAv) (Curatti et al, 2006), K. oxytoca (NifBKo) (Zhao et al, 2007), Clostridium acetobutylicum (NifBCa) (Chen et al, 2001; Wiig et al, 2011), M. infernus (NifBMi) (Wilcoxen et al, 2016), Methanosarcina acetivorans (NifBMa) (Fay et al, 2015), Methanobacterium thermoautotrophicum (NifBMt) (Fay et al, 2015), and C. tepidum (NifBCt, this work), a number of conserved motifs were identified in the S-adenosyl methionine (SAM)-radical domain including an HPC motif, the AdoMet motif (Cx3Cx2C) common to all SAM-radical proteins, an ExRP motif, an AGPG motif, a TxTxN motif and a Cx2CRxDAxG motif
Summary
Nitrogen is abundant on Earth, most of it is in the form of dinitrogen (N≡N or N2). N2 fixing organisms (diazotrophs) capable of converting N2 into NH3, an accessible form of nitrogen, probably appeared in the primordial Earth when the levels of combined nitrogen gradually depleted (Raymond et al, 2004; Canfield et al, 2010). Evolution and fine-tuning of biological nitrogen fixation (BNF) had an immense impact on the Earth’s nitrogen cycle and allowed life to prosper, only a few bacteria and archaea are capable of performing it (Boyd and Peters, 2013). The enzymes that catalyze N2 fixation are called nitrogenases (Burris and Roberts, 1993). All diazotrophs carry the Mo-nitrogenase and may or may not carry
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
