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Flavodoxins are small flavin mononucleotide (FMN)-containing proteins that mediate a variety of electron transfer processes. The primary sequence of flavodoxin from Fusobacterium nucleatum, a pathogenic oral bacterium, is marked with a number of distinct features including a glycine to lysine (K13) substitution in the highly conserved phosphate-binding loop (T/S-X-T-G-X-T), variation in the aromatic residues that sandwich the FMN cofactor, and a more even distribution of acidic and basic residues. The E ox/sq (oxidized/semiquinone; −43 mV) and E sq/hq (semiquinone/hydroquinone; −256 mV) are the highest recorded reduction potentials of known long-chain flavodoxins. These more electropositive values are a consequence of the apoprotein binding to the FMN hydroquinone anion with~70-fold greater affinity compared to the oxidized form of the cofactor. Inspection of the FnFld crystal structure revealed the absence of a hydrogen bond between the protein and the oxidized FMN N5 atom, which likely accounts for the more electropositive E ox/sq . The more electropositive E sq/hq is likely attributed to only one negatively charged group positioned within 12 Å of the FMN N1. We show that natural substitutions of highly conserved residues partially account for these more electropositive reduction potentials.

A number of species within the Fusobacteriaceae family of Gram-negative bacteria uniquely encode for an ornithine decarboxylase/arginase (ODA) that ostensibly channels l-ornithine generated by hydrolysis of l-arginine to putrescine formation. However, two aspartate residues required for coordination to a catalytically obligatory manganese cluster of arginases are substituted for a serine and an asparagine. Curiously, these natural substitutions occur only in a clade of Fusobacterium species that inhabit the oral cavity. Herein, we expressed and isolated full-length ODA from the opportunistic oral pathogen Fusobacterium nucleatum along with the individual arginase and ornithine decarboxylase components. The crystal structure of the arginase domain reveals that it adopts the classical α/β arginase-fold, but metal ions are absent in the active site. As expected, the ureohydrolase activity with l-arginine was not detected for wild-type ODA or the isolated arginase domain. However, engineering of the complete metal coordination environment through site-directed mutagenesis restored Mn2+ binding capacity and arginase activity, although the catalytic efficiency for l-arginine was low (60–100 M–1 s–1). Full-length ODA and the isolated ODC component were able to decarboxylate both l-ornithine and l-arginine to form putrescine and agmatine, respectively, but k cat/K M of l-ornithine was ∼20-fold higher compared to l-arginine. We discuss environmental conditions that may have led to the natural selection of an inactive arginase in the oral associated species of Fusobacterium.

ETV6 is an E 26 t ransformation s pecific family transcriptional repressor that self-associates by its PNT domain to facilitate cooperative DNA binding. Chromosomal translocations frequently generate constitutively active oncoproteins with the ETV6 PNT domain fused to the kinase domain of one of many protein tyrosine kinases. Although an attractive target for therapeutic intervention, the propensity of the ETV6 PNT domain to polymerize via the tight head-to-tail association of two relatively flat interfaces makes it challenging to identify suitable small molecule inhibitors of this protein–protein interaction. Herein, we provide a comprehensive biophysical characterization of the ETV6 PNT domain interaction interfaces to aid future drug discovery efforts and help define the mechanisms by which its self-association mediates transcriptional repression. Using NMR spectroscopy, X-ray crystallography, and molecular dynamics simulations, along with amide hydrogen exchange measurements, we demonstrate that monomeric PNT domain variants adopt very stable helical bundle folds that do not change in conformation upon self-association into heterodimer models of the ETV6 polymer. Surface plasmon resonance–monitored alanine scanning mutagenesis studies identified hot spot regions within the self-association interfaces. These regions include both central hydrophobic residues and flanking salt-bridging residues. Collectively, these studies indicate that small molecules targeted to these hydrophobic or charged regions within the relatively rigid interfaces could potentially serve as orthosteric inhibitors of ETV6 PNT domain polymerization.

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