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A crystallographic study of Cys69Ala flavodoxin II from Azotobacter vinelandii: structural determinants of redox potential.

ABSTRACT: Flavodoxin II from Azotobacter vinelandii is a "long-chain" flavodoxin and has one of the lowest E1 midpoint potentials found within the flavodoxin family. To better understand the relationship between structural features and redox potentials, the oxidized form of the C69A mutant of this flavodoxin was crystallized and its three-dimensional structure determined to a resolution of 2.25 A by molecular replacement. Its overall fold is similar to that of other flavodoxins, with a central five-stranded parallel beta-sheet flanked on either side by alpha-helices. An eight-residue insertion, compared with other long-chain flavodoxins, forms a short 3(10) helix preceding the start of the alpha3 helix. The flavin mononucleotide (FMN) cofactor is flanked by a leucine on its re face instead of the more conserved tryptophan, resulting in a more solvent-accessible FMN binding site and stabilization of the hydroquinone (hq) state. In particular the absence of a hydrogen bond to the N5 atom of the oxidized FMN was identified, which destabilizes the ox form, as well as an exceptionally large patch of acidic residues in the vicinity of the FMN N1 atom, which destabilizes the hq form. It is also argued that the presence of a Gly at position 58 in the sequence stabilizes the semiquinone (sq) form, as a result, raising the E2 value in particular.

SUBMITTER: Alagaratnam S

PROVIDER: S-EPMC2253476 | biostudies-literature | 2005 Sep

REPOSITORIES: biostudies-literature

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A crystallographic study of Cys69Ala flavodoxin II from Azotobacter vinelandii: structural determinants of redox potential.

Alagaratnam Sharmini S van Pouderoyen Gertie G Pijning Tjaard T Dijkstra Bauke W BW Cavazzini Davide D Rossi Gian Luigi GL Van Dongen Walter M A M WM van Mierlo Carlo P M CP van Berkel Willem J H WJ Canters Gerard W GW

Protein science : a publication of the Protein Society 20050901 9

Flavodoxin II from Azotobacter vinelandii is a "long-chain" flavodoxin and has one of the lowest E1 midpoint potentials found within the flavodoxin family. To better understand the relationship between structural features and redox potentials, the oxidized form of the C69A mutant of this flavodoxin was crystallized and its three-dimensional structure determined to a resolution of 2.25 A by molecular replacement. Its overall fold is similar to that of other flavodoxins, with a central five-strand ...[more]

PMID: 16131657

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Project description:The ubiquitous diazotrophic soil bacterium Azotobacter vinelandii has been extensively studied as a model organism for biological nitrogen fixation (BNF). In A. vinelandii, BNF is regulated by the NifL-NifA two-component system, where NifL acts as an antiactivator that tightly controls the activity of the nitrogen fixation-specific transcriptional activator NifA in response to redox, nitrogen, and carbon status. While several studies reported that mutations in A. vinelandii nifL resulted in the deregulation of nitrogenase expression and the release of large quantities of ammonium, knowledge about the specific determinants for this ammonium-excreting phenotype is lacking. In this work, we report that only specific disruptions of nifL lead to large quantities of ammonium accumulated in liquid culture (∼12 mM). The ammonium excretion phenotype is associated solely with deletions of NifL domains combined with the insertion of a promoter sequence in the orientation opposite that of nifLA transcription. We further demonstrated that the strength of the inserted promoter could influence the amounts of ammonium excreted by affecting rnf1 gene expression as an additional requirement for ammonium excretion. These ammonium-excreting nifL mutants significantly stimulate the transfer of fixed nitrogen to rice. This work defines discrete determinants that bring about A. vinelandii ammonium excretion and demonstrates that strains can be generated through simple gene editing to provide promising biofertilizers capable of transferring nitrogen to crops. IMPORTANCE There is considerable interest in the engineering of ammonium-excreting bacteria for use in agriculture to promote the growth of plants under fixed-nitrogen-limiting conditions. This work defines discrete determinants that bring about A. vinelandii ammonium excretion and demonstrates that strains can be generated through simple gene editing to provide promising biofertilizers capable of transferring nitrogen to crops.

Project description:Biological nitrogen fixation, the microbial reduction of atmospheric nitrogen to bioavailable ammonia, represents both a major limitation on biological productivity and a highly desirable engineering target for synthetic biology. However, the engineering of nitrogen fixation requires an integrated understanding of how the gene regulatory dynamics of host diazotrophs respond across sequence-function space of its central catalytic metalloenzyme, nitrogenase. Here, we interrogate this relationship by analyzing the transcriptome of Azotobacter vinelandii engineered with a phylogenetically inferred ancestral nitrogenase protein variant. The engineered strain exhibits reduced cellular nitrogenase activity but recovers wild-type growth rates following an extended lag period. We find that expression of genes within the immediate nitrogen fixation network is resilient to the introduced nitrogenase sequence-level perturbations. Rather the sustained physiological compatibility with the ancestral nitrogenase variant is accompanied by reduced expression of genes that support trace metal and electron resource allocation to nitrogenase. Our results spotlight gene expression changes in cellular processes adjacent to nitrogen fixation as productive engineering considerations to improve compatibility between remodeled nitrogenase proteins and engineered host diazotrophs. IMPORTANCE Azotobacter vinelandii is a key model bacterium for the study of biological nitrogen fixation, an important metabolic process catalyzed by nitrogenase enzymes. Here, we demonstrate that compatibilities between engineered A. vinelandii strains and nitrogenase variants can be modulated at the regulatory level. The engineered strain studied here responds by adjusting the expression of proteins involved in cellular processes adjacent to nitrogen fixation, rather than that of nitrogenase proteins themselves. These insights can inform future strategies to transfer nitrogenase variants to non-native hosts.

Dataset Information

A crystallographic study of Cys69Ala flavodoxin II from Azotobacter vinelandii: structural determinants of redox potential.

Publications

A crystallographic study of Cys69Ala flavodoxin II from Azotobacter vinelandii: structural determinants of redox potential.

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