Biology subjects

Owens, C. P.

Publications and source records attributed to Owens, C. P..

3 recordsLinked to original sources

bioRxiv2023

Mutational analysis of the nitrogenase carbon monoxide protective protein CowN reveals that a conserved C-terminal glutamic acid residue is necessary for its activity

Nitrogenase is the only enzyme that catalyzes the reduction of nitrogen gas into ammonia. Nitrogenase is tightly inhibited by the environmental gas carbon monoxide (CO). Many nitrogen fixing bacteria protect nitrogenase from CO inhibition using the protective protein CowN. This work demonstrates that a conserved glutamic acid residue near CowNs C-terminus is necessary for its function. Mutation of the glutamic acid residue abolishes both CowNs protection against CO inhibition and CowNs ability to bind to nitrogenase. In contrast, a conserved C-terminal cysteine residue is not important for CO protection. Overall, this work uncovers structural features in CowN that are required for its function and provides new insights into its nitrogenase binding and CO protection mechanism.

biochemistry↗

bioRxiv2023

Uncovering structural features that control substrate specificity in a Lactobacillus chlorogenic acid esterase

The structural determinants of chlorogenic acid esterase (CE) substrate specificity are poorly understood. Here, we establish how a Lactobacillus helveticus CE selects for its substrate, chlorogenic acid (CGA). We determine that a Lys residue in an extended loop over the active site imparts substrate specificity by hydrogen bonding to CGA. Mutation of the Lys residue abolishes CGA specificity. Comparison with other bacterial CEs reveals that the extended loop is not conserved. However, the hydrogen bonding functionality to CGA is preserved thanks to other residues. Structural comparison with ferulic acid esterases (FAEs), a related enzyme class, shows that CEs feature a more restricted active site, reflecting the fact that they hydrolyze smaller substrates compared to FAEs.

biochemistry↗

bioRxiv2023

Purification and biochemical characterization of the DNA binding domain of the nitrogenase transcriptional activator NifA from Gluconacetobacter diazotrophicus

NifA is a {sigma}54 activator that turns on bacterial nitrogen fixation under reducing conditions and when fixed cellular nitrogen levels are low. The redox sensing mechanism in -proteobacterial NifA is poorly understood. In this work, we examine if a Cys pair that is part of a C(X)5C motif and located immediately upstream of NifAs DNA binding domain is involved in redox sensing in NifA from the -proteobacterium Gluconacetobacter diazotrophicus (Gd). We hypothesize that the Cys residues redox state may directly influence the DNA binding domains DNA binding affinity and/or alter the proteins 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. The Kd of NC-DBD for its cognate DNA sequence (nifH-UAS) is equal to 20.0 M. The Kd of 2C-DBD for nifH-UAS when the Cys pair is oxidized is 34.5 M. 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 of Gd-NifA does not regulate DNA binding or domain dimerization in a redox dependent manner. This suggests that other Cys residues in NifA, such as those located in the central AAA+ domain, are responsible for redox sensing.

biochemistry↗