Investigation of the vanadium nitrogenase maturation enzymes VnfEN and VnfJ

Abstract: Summary of the dissertation “Investigation of the vanadium nitrogenase maturation enzymes VnfEN and VnfJ” by Jakob Gies-Elterlein
The dissertation deals with maturation enzymes of vanadium nitrogenase from Azotobacter vinelandii. The origin of the carbonate ligand on the FeV cofactor of vanadium nitrogenase, as well as the timing of its insertion in the cluster maturation sequence are unknown.[1] Investigations of the dissertation dealt with 1) VnfEN, which catalyzes the probably last step in cofactor synthesis, as well as 2) an unknown gene, designated vnfJ. The genomic modification of A. vinelandii was established in the Einsle group as an important method for investigation and modification of target genes. Additionally, an elegant system for modification of the vanadium nitrogenase genes vnfDGK and production of the enzyme with an Strep tag were developed.
1) The specificity of VnfEN and NifEN in regard to their individual nitrogenase cluster synthesis pathways could be established through genomic modifications. The investigations revealed the necessity to delete the two other nitrogenases that were not target of the investigations from strains. Especially the negative regulation of the iron only nitrogenase by vanadium was weaker than anticipated. The resulting masking of phenotypes by iron only nitrogenase posed a great challenge to data interpretation by the authors of earlier investigations.[2] This could be avoided by removal of the other nitrogenases. Additionally the studies showed the independence from NifEN and VnfEN for the FeFe-cofactor synthesis.
Heterologous expression in Escherichia coli, as well as expression in A. vinelandii produced VnfEN with a Strep tag. Since crystallization attempts of the enzyme remained unsuccessful, the structure was to be determined by Cryo-EM. The resulting electron density map was not sufficiently well resolved to build a structural model of the enzyme. An AlphaFold prediction did, however, allow for a discussion of the structure. The prediction shows how the cluster could enter into the enzyme and how it would be bound inside of it.
2) Bioinformatic analysis confirmed the presence of vnfJ in diazotrophic organisms and in the exclusive context of vanadium nitrogenases. The deletion of vnfJ resulted in reduced diazotrophic growth in A. vinelandii if based on vanadium nitrogenase. This phenotype was found independent of the stronger effect of deletion of the neighboring gene vnfY.[3] Investigation of vanadium nitrogenase from ΔvnfJ showed reduced nitrogenase activity, but not reduced quantity of the enzyme. This confirmed a maturational and not a regulatory effect of ΔvnfJ.
VnfJ with a C terminal Strep tag could be successfully produced from E. coli. VnfJ did not interact in vitro with mature vanadium nitrogenase. Affinity tag purified vanadium nitrogenase, however, contained small amounts of VnfJ bound in complex. The complex with vanadium nitrogenase could only be obtained by adding E. coli produced, tagged VnfJ to the lysate of ΔvnfJ A. vinelandii cells. Then isolated by re purifying the complex over a Strep tag affinity column. The complex seems to contain only VnfJ and VnfDK, while the additional subunit of the vanadium nitrogenase VnfG is absent.
VnfJ formed protein crystals and the conditions of crystallization could be improved by seeding and fine screening. Se-methionine substituted protein allowed for the experimental phasing and structure solution of VnfJ. The structure has a core of helices, surrounded by flexible loop regions. Docking models show, how the interaction with vanadium nitrogenase could be facilitated. VnfJ probably stabilizes VnfDK until VnfG binds together with insertion of the FeV-cofactor, or even facilitates this binding. Future work on Cryo-EM of the VnfDK J complex might confirm the exact role of VnfJ in maturation of vanadium nitrogenase. Also the question remains of any role of VnfJ for the insertion of the carbonate ligand to FeV cofactor remains to be answered.

[1]: D. Sippel, O. Einsle, Nat Chem Biol 13, 956-960 (2017).
[2]: E. D. Wolfinger, P. E. Bishop, J Bacteriol 173, 7565-7572 (1991).
[3]: C. Ruttimann-Johnson, L. M. Rubio, D. R. Dean, P. W. Ludden, J Bacteriol 185, 2383-2386 (2003)

Angaben zum Objekt

• Standort: Deutsche Nationalbibliothek Frankfurt am Main
• Umfang: Online-Ressource
• Sprache: Englisch
• Anmerkungen: Albert-Ludwigs-Universität Freiburg, Dissertation, 2021

Klassifikation und Themen

• Schlagwort: Nitrogenase ; Stickstofffixierung ; Reaktionsmechanismus ; Dichtefunktionalformalismus ; Azotobacter vinelandii ; Nitrogenase ; Biochemische Methode ; Diazotypie ; Genome Editing ; Proteinbiosynthese ; Mikrobiologe ; Kristallographie ; Strukturmodell ; Röntgendiffraktometrie ; Strukturaufklärung ; Protein-Protein-Wechselwirkung ; Proteindesign ; Metallcluster

Beteiligte, Orts- und Zeitangaben

• Ereignis: Veröffentlichung
• (wo): Freiburg
• (wer): Universität
• (wann): 2023
• Urheber: Gies-Elterlein, Jakob
• Beteiligte Personen und Organisationen: Einsle, Oliver ; Andrade, Susana ; Santos, Patricia C. dos ; Albert-Ludwigs-Universität Freiburg ; Albert-Ludwigs-Universität Freiburg. Fakultät für Chemie und Pharmazie

Weitere Informationen

• DOI: 10.6094/UNIFR/240500
• URN: urn:nbn:de:bsz:25-freidok-2405002
• Rechteinformation: Open Access; Der Zugriff auf das Objekt ist unbeschränkt möglich.
• Letzte Aktualisierung: 15.08.2025, 07:34 MESZ