Protein Protection: Characterizing how CowN Protects Nitrogenase

Nitrogen fixation occurs in two major processes, the industrial haber bosch process and fixation via a biological enzyme called nitrogenase. The haber bosch process is how most nitrogen used in agriculture is converted into ammonia. However, one major drawback is that this process requires a lot of fossil fuels and is thereby not an environmentally friendly process. With nitrogenase, the enzyme converts dinitrogen into ammonia using biological energy in the form of ATP, making nitrogen fixation a more biologically friendly process. Carbon Monoxide is known to inhibit the function of nitrogenase, meaning that under conditions where CO is present, nitrogen fixation is unable to occur. In order to prevent CO inhibition, cells containing nitrogenase must find a way to avoid these inhibitory conditions. It was found that Nitrogenase seems to be protected by another protein, CowN. This poster describes the mechanism by which CowN protects Nitrogenase. Specifically, CowN binds to either the entrance to a proposed CO channel or near the active site of nitrogenase. Both potential CowN binding locations could prevent CO from reaching the active site and therefore enable nitrogenase to avoid inhibition by CO

Purification and Biochemical Characterization of the DNA Binding Domain of the Nitrogenase Transcriptional Activator NifA from \u3cem\u3eGluconacetobacter diazotrophicus\u3c/em\u3e

NifA is a σ54 activator 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-proteobacterium Gluconacetobacter 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. The Kd of NC-DBD for its cognate DNA sequence (nifH-UAS) is equal to 20.0 µM. The Kd of 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 of Gd-NifA does not regulate DNA binding or domain dimerization in a redox dependent manner

Calmodulin’s Interaction with α- Synuclein, a Protein Implicated in Parkinson’s Disease

60,000 people in one year diagnosed, 1 million in the United States, and over 10 million worldwide have Parkinson’s disease (PD), which is the 2nd most common neurodegenerative disease. PD is prevalent in males and is typically seen in patients in their 60s. The most notable symptom of PD is the degeneration of neuronal control, especially in the hands. Over $156 million is spent on researching this disease and about$25 billion is spent for diagnosed patients each year. Aside from managing the financial burdens of PD, patients also have physical burdens. Most patients develop tremors and have difficulties writing, eating, and can degenerate quickly. The PD has previously been attributed to the lack of the neurotransmitter, dopamine, in the patient’s brain; however recent biochemical studies have surfaced other biomolecular mechanisms that attribute to PD, such as the interaction between Calmodulin and α-Synuclein. Calmodulin (CaM) is a protein found in the brain of healthy patients and is an intermediate calcium (Ca2+) binding messenger with over 100 different targets in eukaryotic cells. α- Synuclein (α-Syn) is a protein found at the ends of neurons in the presynaptic terminals in healthy patients, suggesting involvement with neurotransmitter signaling, however the exact function of α- Syn is still under investigation. Recent studies show that α- Syn and CaM interact resulting in protein aggregation. The α- Syn aggregation is the main structural component of Lewy bodies which is enhanced in the presence of Ca2+. Lewy bodies are known to develop in cranial nerve cells of PD patients and interrupts neuronal function. Little is known about how Lewy bodies attribute to abnormalities in PD patients, but there are connections to low levels of acetylcholine and dopamine, in addition to an interruption of signals between nerve cells. By using fluorescence spectroscopy, we studied the interaction between α- Syn and CaM and explore Ca2+’s role in the interaction that promotes the degenerateness of PD patients

Hydrolysis of Chlorogenic Acid in Sunflower Flour Increases Consumer Acceptability of Sunflower Flour Cookies by Improving Cookie Color

Sunflower meal, a byproduct of sunflower oil pressing, is not commonly used in alkaline baking applications. This is because chlorogenic acid, the main phenolic antioxidant in sunflower seeds, reacts with protein, giving the baked product a green discoloration. Our group previously demonstrated that a chlorogenic acid esterase from Lactobacillus helveticus hydrolyzes chlorogenic acid in sunflower dough cookie formulations, resulting in cookies that were brown instead of green. This study presents a sensory analysis to determine the acceptability of enzymatically upcycled sunflower meal as an alternative protein source for those allergic to meals from legumes or tree nuts. We hypothesized that the mechanism of esterase-catalyzed chlorogenic acid breakdown does not influence the cookies’ sensory properties other than color and that consumers would prefer treated, brown cookies over non-treated cookies. Cookies made from sunflower meal were presented under green lights to mask color and tested by 153 panelists. As expected, the sensory properties (flavor, smell, texture, and overall acceptability) of the treated and non-treated cookies were not statistically different. These results corroborate proximate analysis, which demonstrated that there was no difference between enzymatically treated and non-treated cookies other than color and chlorogenic acid content. After the cookie color was revealed, panelists strongly preferred the treated cookies with 58% indicating that they “probably” or “definitely” would purchase the brown cookies, whereas only 5.9% would buy green, non-treated cookies. These data suggest that esterase-catalyzed breakdown of chlorogenic acid represents an effective strategy to upcycle sunflower meal for baking applications

Preventing Chlorogenic Acid Quinone-Induced Greening in Sunflower Cookies by Chlorogenic Acid Esterase and Thiol-based Dough Conditioners

Sunflower seeds contain a high concentration of chlorogenic acid (CGA), which reacts with amino acids to form green pigments under alkaline conditions during food processing. Here, we present two approaches to prevent green pigment formation in sunflower cookies by (A) Addition of free thiols from cysteine and glutathione to sunflower cookie dough and (B) hydrolyzing CGA into caffeic acid and quinic acid with a CGA esterase from Lactobacillus helveticus. Greening occurred more slowly with cysteine; however, neither cysteine nor glutathione prevented greening in the cookies during storage. Chlorogenic acid esterase hydrolyzed CGA in both sunflower butter and flour, resulting in the complete elimination of greening in the sunflower cookies. CGA esterase treatment was efficient as the enzyme could be applied in low amounts (\u3c100 ppm) directly to the dough without needing to pretreat either sunflower butter or flour. Overall, our data indicate that CGA esterase treatment was an effective method of eliminating unwanted greening in sunflower cookies made with baking soda. Long term, these results may represent a method of increasing the use of sunflower butter and flour in high pH baking applications by enabling their use in neutrally colored baked products such as cookies and muffins

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 the C-terminus of Gluconacetobacter diazotrophicus CowN is necessary for its function. Mutation of the glutamic acid residue abolishes both CowN’s protection against CO inhibition and the ability of CowN to bind to nitrogenase. In contrast, a conserved C-terminal cysteine residue is not important for CO protection by CowN. 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

The Structure of a \u3cem\u3eLactobacillus helveticus\u3c/em\u3e Chlorogenic Acid Esterase and the Dynamics of Its Insertion Domain Provide Insights into Substrate Binding

Chlorogenic acid esterases (ChlEs) are a useful class of enzymes that hydrolyze chlorogenic acid (CGA) into caffeic and quinic acid. 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 from Lactobacillus helveticus (Lh). Molecular dynamics simulations suggest that substrate access to the active site of LhChlE is modulated by two hairpin loops above the active site. Docking simulations and mutational analysis suggest that two residues within the loops, Gln145 and Lys164, are important for CGA binding. Lys164 provides a slight substrate preference for CGA, whereas Gln145 is required for efficient turnover. This work is the first to examine the dynamics of a bacterial ChlE and provides insights on the substrate binding preference and turnover in this type of enzyme

A Highly Active Esterase from \u3cem\u3eLactobacillus helveticus\u3c/em\u3e Hydrolyzes Chlorogenic Acid in Sunflower Meal to Prevent Chlorogenic Acid Induced Greening in Sunflower Protein Isolates

Chlorogenic acid (CGA) is an ester between caffeic and quinic acid. It is found in many foods and reacts with free amino groups in proteins at alkaline pH, leading to the formation of an undesirable green pigment in sunflower seed-derived ingredients. This paper presents the biochemical characterization and application of a highly active chlorogenic acid esterase from Lactobacillus helveticus. The enzyme is one of the most active CGA esterases known to date with a Km of 0.090 mM and a kcat of 82.1 s−1. The CGA esterase is easily expressed recombinantly in E. coli in large yields and is stable over a wide range of pH and temperatures. We characterized CGA esterase’s kinetic properties in sunflower meal and demonstrated that the enzyme completely hydrolyzes CGA in the meal. Finally, we showed that CGA esterase treatment of sunflower seed meal enables the production of pale brown sunflower protein isolates using alkaline extraction. This work will allow for more widespread use of sunflower-derived products in applications where neutrally-colored food products are desired

CowN Sustains Nitrogenase Turnover in the Presence of the Inhibitor Carbon Monoxide

Nitrogenase is the only enzyme capable of catalyzing nitrogen fixation, the reduction of dinitrogen gas (N2) to ammonia (NH3). Nitrogenase is tightly inhibited by the environmental gas carbon monoxide (CO). Nitrogen-fixing bacteria rely on the protein CowN to grow in the presence of CO. However, the mechanism by which CowN operates is unknown. Here, we present the biochemical characterization of CowN and examine how CowN protects nitrogenase from CO. We determine that CowN interacts directly with nitrogenase and that CowN protection observes hyperbolic kinetics with respect to CowN concentration. At a CO concentration of 0.001 atm, CowN restores nearly full nitrogenase activity. Our results further indicate that CowN’s protection mechanism involves decreasing the binding affinity of CO to nitrogenase’s active site approximately tenfold without interrupting substrate turnover. Taken together, our work suggests CowN is an important auxiliary protein in nitrogen fixation that engenders CO tolerance to nitrogenase

Differential Function of Lip Residues in the Mechanism and Biology of an Anthrax Hemophore

To replicate in mammalian hosts, bacterial pathogens must acquire iron. The majority of iron is coordinated to the protoporphyrin ring of heme, which is further bound to hemoglobin. Pathogenic bacteria utilize secreted hemophores to acquire heme from heme sources such as hemoglobin. Bacillus anthracis, the causative agent of anthrax disease, secretes two hemophores, IsdX1 and IsdX2, to acquire heme from host hemoglobin and enhance bacterial replication in iron-starved environments. Both proteins contain NEAr-iron Transporter (NEAT) domains, a conserved protein module that functions in heme acquisition in Gram-positive pathogens. Here, we report the structure of IsdX1, the first of a Gram-positive hemophore, with and without bound heme. Overall, IsdX1 forms an immunoglobin-like fold that contains, similar to other NEAT proteins, a 310-helix near the heme-binding site. Because the mechanistic function of this helix in NEAT proteins is not yet defined, we focused on the contribution of this region to hemophore and NEAT protein activity, both biochemically and biologically in cultured cells. Site-directed mutagenesis of amino acids in and adjacent to the helix identified residues important for heme and hemoglobin association, with some mutations affecting both properties and other mutations affecting only heme stabilization. IsdX1 with mutations that reduced the ability to associate with hemoglobin and bind heme failed to restore the growth of a hemophore-deficient strain of B. anthracis on hemoglobin as the sole iron source. These data indicate that not only is the 310-helix important for NEAT protein biology, but also that the processes of hemoglobin and heme binding can be both separate as well as coupled, the latter function being necessary for maximal heme-scavenging activity. These studies enhance our understanding of NEAT domain and hemophore function and set the stage for structure-based inhibitor design to block NEAT domain interaction with upstream ligands