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Abstract NifA is a σ⁵⁴activator that turns on bacterial nitrogen fixation under reducing conditions and when fixed cellular nitrogen levels are low. The redox sensing mechanism in NifA is poorly understood. In α- and β-proteobacteria, redox sensing involves two pairs of Cys residues within and immediately following the protein’s central AAA⁺domain. In this work, we examine if an additional Cys pair that is part of a C(X)₅ C motif and located immediately upstream of the DNA binding domain of NifA from the α-proteobacteriumGluconacetobacter diazotrophicus(Gd) is involved in redox sensing. We hypothesize that the Cys residues’ redox state may directly influence the DNA binding domain’s DNA binding affinity and/or alter the protein’s oligomeric sate. Two DNA binding domain constructs were generated, a longer construct (2C-DBD), consisting of the DNA binding domain with the upstream Cys pair, and a shorter construct (NC-DBD) that lacks the Cys pair. TheK_dof NC-DBD for its cognate DNA sequence (nifH-UAS) is equal to 20.0 µM. TheK_dof 2C-DBD for nifH-UAS when the Cys pair is oxidized is 34.5 µM. Reduction of the disulfide bond does not change the DNA binding affinity. Additional experiments indicate that the redox state of the Cys residues does not influence the secondary structure or oligomerization state of the NifA DNA binding domain. Together, these results demonstrate that the Cys pair upstream of the DNA binding domain ofGd-NifA does not regulate DNA binding or domain dimerization in a redox dependent manner. 

Chlorogenic acid esterases (ChlEs) are a useful class of enzymes that hydrolyze chlorogenic acid (CGA) into caffeic and quinic acids. ChlEs can break down CGA in foods to improve their sensory properties and release caffeic acid in the digestive system to improve the absorption of bioactive compounds. This work presents the structure, molecular dynamics, and biochemical characterization of a ChlE fromLactobacillus helveticus(Lh). Molecular dynamics simulations suggest that substrate access to the active site ofLhChlE is modulated by two hairpin loops above the active site. Docking simulations and mutational analysis suggest that two residues within the loops, Gln₁₄₅and Lys₁₆₄, are important for CGA binding. Lys₁₆₄provides a slight substrate preference for CGA, whereas Gln₁₄₅is required for efficient turnover. This work is the first to examine the dynamics of a bacterial ChlE and provides insights on substrate binding preference and turnover in this type of enzyme.