Nonpolar Amino Acids Research Articles

In-silico Characterization of ICE1 Transcriptional Factor in Cold Signalling Network in Mungbean (Vigna radiata L.)

Aim: Mungbean (Vigna radiata L.) is a leguminous crop with high rich of protein and susceptible to cold stress. In silico characterization of ICE1, a cold tolerant transcriptional factor in Vigna radiata and transcriptional regulatory network with other species of Vigna and with special refrence to legume crops.
 Methodology: Genomic sequences of Vigna radiata, V. angularis and V. unguculata and other leguminous crops were retrieved from NCBI (https://pubmed.ncbi.nlm.nih.gov) and used for multiple sequence alignment, Phylogenetic and comparative analysis of ICE1 protein and motif analysis.
 Results: The present study showed that Vigna radiata ICE1 (VrICE1) gene was closely related to VaICE1 gene (Vigna angularis) and then VuICE1 gene (Vigna unguiculata). The study also revealed that the bHLH domain region (247-298aa) was the low complexity region and co-localization signals of V.radiataICE1. There were a similarities and dissimilarities of the ICE1 protein isoforms in V.radiata. It was noted that VrICE1 had a average of hydropathicity (GRAVY) of -0.593 in one and -0.6 in other. An instability index of 64.58 and 64.90. VrICE1 had a higher percentage of non-polar amino acid content and more number of random coils followed by alpha helix in secondary structure. The study also revealed that there was major involvement of VrICE1 proteins in biological and molecular functions under cold stress.
 Conclusion: The bioinformatics tools help researchers in getting information with regard to the functional aspect of the gene with respect to cold tolerance. A comprehensive analysis of the different physical and chemical properties of Vigna species could help us to identify their diversified usefulness.

Aquatic toxicity of tire microplastics on marine and freshwater organisms: An in silico approach

Tire wear particles are a notable source of tire microplastics (TMPs) in the environment. However, only a few reports have focused on the aquatic toxicity effects of composite TMPs and their additives and the mechanistic analysis at the microscopic level. Therefore, this paper study the toxic effects of tire microplastics and their additives on zebrafish based on theoretical chemical calculation method (Taguchi orthogonal experiment method, full factorial experimental design, molecular docking, and molecular dynamics computational technique). We designed five kinds of proportioning schemes of tire rubber polymers and additive components (64 groups in each). The compound toxicity effects of the tire rubber polymers and their additives on zebrafish were simulated and calculated. The simulation results indicated styrene butadiene rubber had the most significant toxic effect on zebrafish. Subsequently, taking the composition ratio scheme of styrene butadiene rubber with the lowest biotoxicity effect as an example, we analyzed the main effects, second-order interactions, and third-order interactions of styrene butadiene rubber polymer and its additive combination in terms of biotoxicity using the fixed effects model. The toxic effects (developmental toxicity, neurotoxicity, and reproductive toxicity) of styrene butadiene rubber on marine and freshwater organisms could be drastically alleviated by adjusting the ratio of rubber additives. The analysis of the interaction between amino acid residues and non-bonds during the docking process of styrene butadiene rubber and toxic receptors revealed the interaction mechanisms between the styrene butadiene rubber polymer and its additives and between the additive molecules. Hydrophobic interaction was found to be the key factor for the binding of styrene butadiene rubber additives to nonpolar amino acids in the receptor proteins. Our findings are expected to provide theoretical support for identifying and regulating the toxicity characteristics of rubber TMPs and to aid in proposing a strategy to alleviate the toxic effects on aquatic organisms.

Structural and functional modification of proteins from black soybean Aquasoya via ultrasonication.

Plant-based proteins obtained from agricultural by-products have garnered growing interest in response to consumer awareness of health and environmental issues. This study aimed to improve the functionalities of the proteins recovered from black soybean Aquasoya (PBSA) by modifying their structure via ultrasonication. PBSA was ultrasonicated with a frequency of 40kHz at 350W for different time periods (0, 20, 40, and 60min), and its structural characteristics, physicochemical properties, and functional properties were investigated. Ultrasonication left the primary structure intact but altered the secondary and tertiary structures of the PBSA; α-helix and β-sheet contents decreased, random coil contents increased, and buried non-polar amino acid residues were exposed. Moreover, ultrasound promoted an increase in free sulfhydryl content and a reduction in particle size. Consequently, functional properties, such as solubility, emulsion stability, and foaming performance were improved by modifying the structure and physicochemical properties of PBSA. This work demonstrates the potential of ultrasound, which is favorable for modifying the spatial conformation and related functionalities of proteins, thus meeting the needs of manufacturers to use function-enhanced proteins as food additives.

Sequence Properties of An Intramolecular Interaction That Inhibits p53 DNA Binding.

An intramolecular interaction between the p53 transactivation and DNA binding domains inhibits DNA binding. To study this autoinhibition, we used a fragment of p53, referred to as ND WT, containing the N-terminal transactivation domains (TAD1 and TAD2), a proline rich region (PRR), and the DNA binding domain (DBD). We mutated acidic, nonpolar, and aromatic amino acids in TAD2 to disrupt the interaction with DBD and measured the effects on DNA binding affinity at different ionic strengths using fluorescence anisotropy. We observed a large increase in DNA binding affinity for the mutants consistent with reduced autoinhibition. The ΔΔG between DBD and ND WT for binding a consensus DNA sequence is -3.0 kcal/mol at physiological ionic strength. ΔΔG increased to -1.03 kcal/mol when acidic residues in TAD2 were changed to alanine (ND DE) and to -1.13 kcal/mol when all the nonpolar residues, including W53/F54, were changed to alanine (ND NP). These results indicate there is some cooperation between acidic, nonpolar, and aromatic residues from TAD2 to inhibit DNA binding. The dependence of DNA binding affinity on ionic strength was used to predict excess counterion release for binding both consensus and scrambled DNA sequences, which was smaller for ND WT and ND NP with consensus DNA and smaller for scrambled DNA overall. Using size exclusion chromatography, we show that the ND mutants have similar Stokes radii to ND WT suggesting the mutants disrupt autoinhibition without changing the global structure.

Significantly improved detection performances of immunoassay for ractopamine in urine based on highly urea-tolerant rabbit monoclonal antibody.

Highly sensitive and accurate screening of ractopamine (RAC) residue in animal urine is greatly needed to ensure food security. The detection performance of immunoassay for RAC was always seriously harmed by the antibody inactivation derived from urea. Here, we first discovered one rabbit monoclonal antibody (RmAb) to RAC with a high affinity of 0.007ngmL-1 and a surprising urea tolerance of 3M urea, which is beneficial for developing robustly developed immunoassay in urine without sample pretreatment. The limits of detection of developed indirect competitive enzyme-linked immunosorbent assay based on RmAb1 for RAC were 0.0042-0.014μgL-1 with the coefficient of variation below 11.7% in swine, sheep, and cow urine, significantly improved 10-100-fold in sensitivity. Moreover, the urea-tolerant mechanism of RmAb1 showed that more non-polar amino acids, more hydrogen bond donors on the surface, and preponderant Pi interaction of antibody-RAC all contributed to the stability of the RmAb1 in a high concentration of urea.

The preparation and antioxidant activities of four 2-aminoacyl-chitooligosaccharides

Chitooligosaccharides (COS) with two different molecular weights are acylated with four nonpolar amino acids: glycine (Gly), alanine (Ala), valine (Val) and leucine (Leu) to obtain 2-aminoacetyl-chitooligosaccharide (2-GlyCOS), 2-aminopropionyl-chitooligosaccharide (2-AlaCOS), 2-amino-3-methylbutyryl-chitooligosaccharide (2-ValCOS), and 2-amino-4-methylpentanoyl-chitooligosaccharide (2-LeuCOS). The structure of the derivatives was characterized by FT-IR spectroscopy, 13C NMR spectroscopy, and elemental analysis. The antioxidant activities of the derivatives, such as hydroxyl radical (·OH) scavenging ability, superoxide anion (O2·-) scavenging ability, reducing ability, and DPPH radical scavenging ability, were investigated using various established systems. Compared with chitooligosaccharide and nonpolar amino acids, all derivatives have strong scavenging ability toward hydroxyl radicals and superoxide anions, and the clearance rate was 19.05% and 67.70% separately. The reducing ability and DPPH free radical scavenging ability of the derivatives are only 0.021Abs and 32.97%. Among them, only 2-AlaLCOS has significant reducing ability, and the value can reach 0.143Abs. The above results showed that the antioxidant activity of some derivatives was higher than that of chitooligosaccharide. The water solubility of the new derivatives was also greatly improved compared to that of nonpolar amino acids. Therefore, the application of 2-aminoacyl-chitooligosaccharides (2-AACOS) in antioxidants has laid a foundation and has certain potential application value in the fields of medicine, agriculture, and animal husbandry.

Identification of amino acid residues in the MT-loop of MT1-MMP critical for its ability to cleave low-density lipoprotein receptor

Low-density lipoprotein receptor (LDLR) mediates clearance of plasma LDL cholesterol, preventing the development of atherosclerosis. We previously demonstrated that membrane type 1-matrix metalloproteinase (MT1-MMP) cleaves LDLR and exacerbates the development of atherosclerosis. Here, we investigated determinants in LDLR and MT1-MMP that were critical for MT1-MMP-induced LDLR cleavage. We observed that deletion of various functional domains in LDLR or removal of each of the five predicted cleavage sites of MT1-MMP on LDLR did not affect MT1-MMP-induced cleavage of the receptor. Removal of the hemopexin domain or the C-terminal cytoplasmic tail of MT1-MMP also did not impair its ability to cleave LDLR. On the other hand, mutant MT1-MMP, in which the catalytic domain or the MT-loop was deleted, could not cleave LDLR. Further Ala-scanning analysis revealed an important role for Ile at position 167 of the MT-loop in MT1-MMP’s action on LDLR. Replacement of Ile167 with Ala, Thr, Glu, or Lys resulted in a marked loss of the ability to cleave LDLR, whereas mutation of Ile167 to a non-polar amino acid residue, including Leu, Val, Met, and Phe, had no effect. Therefore, our studies indicate that MT1-MMP does not require a specific cleavage site on LDLR. In contrast, an amino acid residue with a hydrophobic side chain at position 167 in the MT-loop is critical for MT1-MMP-induced LDLR cleavage.

Effect of N-glycosylation on secretion, stability, and biological activity of recombinant human interleukin-3 (hIL-3) in Pichia pastoris.

Human interleukin-3 (hIL-3) is a clinically important cytokine used to treat hematological malignancies, bone marrow transplantation, cytopenias, and immunological disorders. The cloning of hIL-3 gene was previously reported by our group, where its expression was optimized under methanol-inducible AOX1 promoter having N-terminal α mating factor signal sequence from Saccharomyces cerevisiae. This study investigated the role of glycosylation pattern on its molecular stability, secretion efficiency, and biological activity using the mutagenesis approach. The two N-linked glycosylation positions at N15th (Asn15) and N70th (Asn70) were sequentially mutated to generate three recombinant hIL-3 variants, i.e., N15A, N70A, and N15/70A. Asparagine at these positions was replaced with non-polar alanine amino acid (Ala, A). The alteration of N-linked glycosylation sites was disadvantageous to its efficient secretion in Pichia pastoris, where a 52.32%, 36.48%, 71.41%lower production was observed in N15A, N70A, and N15/70A mutants, respectively, as compared to native control. The fully glycosylated native hIL-3 protein showed higher thermal stability over its deglycosylated counterparts. The biological activity of native, N15A, N70A, and N15/70A hIL-3 protein was evaluated, where N15/70A mutant showed slightly higher proliferation efficacy than other combinations.

Study of key amino acid residues of GH66 dextranase for producing high-degree polymerized isomaltooligosaccharides and improving of thermostability.

Obtaining high-degree polymerized isomaltose is more difficult while achieving better prebiotic effects. We investigated the mutation specificity and enzymatic properties of SP5-Badex, a dextranase from the GH66 family of Bacillus aquimaris SP5, and determined its mutation sites through molecular docking to obtain five mutants, namely E454K, E454G, Y539F, N369F, and Y153N. Among them, Y539F and Y153N exhibited no enzymatic activity, but their hydrolysates included isomaltotetraose (IMO4). The enzymatic activity of E454G was 1.96 U/ml, which was 3.08 times higher than that before mutation. Moreover, 70% of the enzymatic activity could be retained after holding at 45°C for 180 min, which was 40% higher than that of SP5-Badex. Furthermore, its IMO4 content was 5.62% higher than that of SP5-Badex after hydrolysis at 30°C for 180 min. To investigate the effect of different amino acids on the same mutation site, saturation mutation was induced at site Y153, and the results showed that the enzyme activity of Y153W could be increased by 2 times, and some of the enzyme activity could still be retained at 50°C. Moreover, the enzyme activity increased by 50% compared with that of SP5-Badex after holding at 45°C for 180 min, and the IMO4 content of Y153W was approximately 64.97% after hydrolysis at 30°C for 180 min, which increased by approximately 12.47% compared with that of SP5-Badex. This site is hypothesized to rigidly bind to nonpolar (hydrophobic) amino acids to improve the stability of the protein structure, which in turn improves the thermal stability and simultaneously increases the IMO4 yield.

Taste peptides derived from Stropharia rugosoannulata fermentation mycelium and molecular docking to the taste receptor T1R1/T1R3.

This study identified the peptides in the fermentation mycelia of Stropharia rugosoannulata. The molecular weight of the peptides was below 3,000 Da. Heptapeptides to decapeptides were the main peptides in the fermentation mycelia of S. rugosoannulata. More than 50% of the peptides had salty and umami taste characteristics, and the long-chain peptides (decapeptides to 24 peptides) also played an essential role in the pleasant taste characteristics of mycelium. In the salty and umami peptide of S. rugosoannulata, the distribution of non-polar hydrophobic amino acids and polar-uncharged amino acids accounted for a relatively high proportion, and the proportion of polar-uncharged amino acids further increased, with the extension of the peptide chain. P, F, I, l, V, G, S, T, and D were the amino acids with a high proportion in the peptides. The taste peptides can bind to more than 60% of the active amino acid residues in the cavity-binding domain of the T1R1/T1R3 receptors. Hydrogen bond interaction was the primary mode of interaction between the peptides and the receptor. The first and second amino acid residues (such as S, V, E, K, G, and A) at the C-terminal and N-terminal of the peptides were easy to bind to T1R1/T1R3 receptors. Asp108, Asn150, Asp147, Glu301, Asp219, Asp243, Glu70, Asp218 in T1R1, and Glu45, Glu148, Glu301, Glu48, and Ala46 in TIR3 were the key active amino acid sites of taste peptides binding to T1R1/T1R3 receptors.

A Computational-Experimental Investigation of the Molecular Mechanism of Interleukin-6-Piperine Interaction.

Herein, we elucidate the biophysical aspects of the interaction of an important protein, Interleukin-6 (IL6), which is involved in cytokine storm syndrome, with a natural product with anti-inflammatory activity, piperine. Despite the role of piperine in the inhibition of the transcriptional protein NF-κB pathway responsible for activation of IL6 gene expression, there are no studies to the best of our knowledge regarding the characterisation of the molecular interaction of the IL6-piperine complex. In this context, the characterisation was performed with spectroscopic experiments aided by molecular modelling. Fluorescence spectroscopy alongside van’t Hoff analyses showed that the complexation event is a spontaneous process driven by non-specific interactions. Circular dichroism aided by molecular dynamics revealed that piperine caused local α-helix reduction. Molecular docking and molecular dynamics disclosed the microenvironment of interaction as non-polar amino acid residues. Although piperine has three available hydrogen bond acceptors, only one hydrogen-bond was formed during our simulation experiments, reinforcing the major role of non-specific interactions that we observed experimentally. Root mean square deviation (RMSD) and hydrodynamic radii revealed that the IL6-piperine complex was stable during 800 ns of simulation. Taken together, these results can support ongoing IL6 drug discovery efforts.

Impacts of perfluorooctanesulfonic acid on plant biometrics and grain metabolomics of wheat (Triticum aestivum L.)

Per- and polyfluoroalkyl substances (PFAS) are utility chemicals that have become environmental contaminants of global concern due to their bioaccumulative and persistent nature. The accumulation and effects of perfluorooctanesulfonic acid (PFOS), a type of PFAS, on agricultural plants is a serious environmental concern that needs to be investigated. Wheat was exposed to soil amended with 0, 25, and 50 mg/kg PFOS and assessed for PFOS’ impacts via agronomic measurements, biochemical assays, PFOS uptake, and ionomics and metabolomics analyses over the lifetime of the plant. PFOS was taken up in the roots and relocated to the grains, which accumulated PFOS at levels 12-18 times higher in treated plants than in control. Compared to control, 50 mg/kg PFOS reduced chlorophyll content by 49% and root biomass by 37% early on during the exposure. At the end of full life cycle exposure, PFOS did not affect the agronomic and productivity status of plants. However, PFOS impacted grain quality by reducing concentrations of macroelements (Mg, K, and P) and levels of important sugar metabolites (e.g., sucrose, glucose 6-phosphate, fructose 6-phosphate, trehalose). PFOS also decreased the abundance of non-polar proteinogenic amino acids but increased the levels of polar amino acids in grains. These findings highlight impacts of PFOS on an important agronomic crop.

Interactions of boron nitride nanosheet with amino acids of differential polarity

Free amino acids represent a category of different biomolecules in the blood plasma, which bond together to make up larger organic molecules such as peptides and proteins. Their interactions with biocompatible nanoparticles are especially important for plasma-related biomedical applications. Among the various nanomaterials, the applications of carbon and boron nitride-based nanotubes/nanosheets have shown a huge increase in recent years. The effect of molecular polarity on the interaction between a boron nitride nanosheet (BNNS) and amino acids is investigated with quantum mechanical calculations by density functional theory (DFT), classical MD simulations, and well-tempered metadynamics simulations. Four representative amino acids, namely, alanine (Ala), a nonpolar amino acid, and aspartic acid (Asp), lysine (Lys) and serine (Ser), three polar amino acids are considered for their interactions with BNNS. In DFT calculations, the values of the adsorption energies for Lys-BNNS and Ser-BNNS complexes are − 48.32 and − 32.89 kJ/mol, respectively, which are more stable than the other cases. Besides, the adsorption energy calculated confirms the exergonic reactions for all investigated systems; it implied that the interaction is favorable electronically. The MD results show that the LYS molecules have a higher attraction toward BNNS because of its alkane tail in its side chain, and the ASP revealed the repulsion force originating from its COO– group. All the results are confirmed by free energy analyzes in which the LYS showed the highest adsorption free energy at a relatively farther distance than other complexes. In fact, our results revealed the contribution of functional groups and backbone of the amino acids in the adsorption or repulsion features of the studied systems.

Desalination Potential of Aquaporin-Inspired Functionalization of Carbon Nanotubes: Bridging Between Simulation and Experiment.

Outstanding water/ion selectivity of aquaporins paves the way for bioinspired desalination membranes. Since the amino acid asparagine (Asn) plays a critical role in the fast water conduction of aquaporins through hydrogen bonding interactions, we adapted this feature by functionalizing carbon nanotubes (CNTs) with Asn. We also studied a nonpolar amino acid and carboxylate functional groups for comparison. Computation of the ideal performance of individual CNTs at atomistic scale is a powerful tool for probing the effect of tip-functionalized CNTs on water and ion transport mechanism. Molecular simulation study suggests that steric effects required for ion rejection compromise fast water conductivity; however, an Asn functional group having polarity and hydrogen bonding capability can be used to balance this trade-off to some extent. To test our hypothesis, we incorporated functionalized CNTs (f-CNTs) into the in situ polymerized selective polyamide (PA) layer of thin film nanocomposite membranes and compared their experimental RO desalination performance. The f-CNTs were found to change the separation environment through modification of cross-linking density, thickness, and hydrophilicity of the PA layer. Asn functionalization led to more cross-linked and thinner PA layer while hydrophilicity is improved compared to other functional groups. Accordingly, water permeance is increased by 25% relative to neat PA with a salt rejection above 98%. Starting from the nanomaterial itself and benefiting from molecular simulation, it is possible to design superior membranes suited for practical applications.

Nonpolar hydrophobic amino acids tune the enzymatic activity of lysozyme

We have used five different hydrophobic L-amino acids (Gly, Ala, Val, Leu, Ile) as molecular crowders to investigate their role on the enzymatic activity of lysozyme towards Micrococcus lysodeikticus (M. lys.)cell as substrate. We found that except Ile, all other amino acids show a bell like profile of catalytic efficiency (kcat/Km) with their increasing concentration whereas for Ile, the value is gradually increasing. The trend of activation energy (Ea) is also well correlated with the catalytic efficiency of lysozyme. At low concentration of amino acids, soft interaction predominates whereas at higher concentration range, excluded volume, viscosity, hydrophobicity combinedly decrease the activity of lysozyme.

Effects of phosphorylation pretreatment and subsequent transglutaminase cross-linking on physicochemical, structural, and gel properties of wheat gluten

The presence of a large number of hydrophobic groups and non-polar amino acids in the wheat gluten (WG) is responsible for its poor water solubility, greatly limiting its industrial applications. Our results showed that the solubility and zeta potential of WG were significantly (P < 0.05) improved with the increasing concentration of sodium tripolyphosphate (STP), while the average particle size of WG was decreased. After WG was incubated with TGase, phosphorylation pretreatment significantly increased apparent viscosity of WG dispersant solution, suggesting that phosphorylation treatment promoted the generation of cross-linked polymers. In addition, phosphorylation pretreatment enhanced hydrophobic interactions and disulfide bond formation between TGase-induced WG gels, thus leading to a more homogeneous and dense three-dimensional network structure of gel, which was confirmed by SEM micrographs. To summarize, STP can be used as an effective additive for the modification of WG with an improved degree of TGase-mediated cross-linking for better rheological and gel properties.

Kir7.1 disease mutant T153I within the inner pore affects K+ conduction.

Inward-rectifier potassium channel 7.1 (Kir7.1) is present in the polarized epithelium, including the retinal pigmented epithelium. A single amino acid change at position 153 in the KCNJ13 gene, a substitution of threonine to isoleucine in the Kir7.1 protein, causes blindness. We hypothesized that the disease caused by this single amino acid substitution within the transmembrane protein domain could alter the translation, localization, or ion transport properties. We assessed the effects of amino acid side-chain length, arrangement, and polarity on channel structure and function. We showed that the T153I mutation yielded a full-length protein localized to the cell membrane. Whole cell patch-clamp recordings and chord conductance analyses revealed that the T153I mutant channel had negligible K+ conductance and failed to hyperpolarize the membrane potential. However, the mutant channel exhibited enhanced inward current when rubidium was used as a charge carrier, suggesting that an inner pore had formed and the channel was dysfunctional. Substituting with a polar, nonpolar, or short side-chain amino acid did not affect the localization of the protein. Still, it had an altered channel function due to differences in pore radius. Polar side chains (cysteine and serine) with inner pore radii comparable to wildtype exhibited normal inward K+ conductance. Short side chains (glycine and alanine) produced a channel with wider than expected inner pore size and lacked the biophysical characteristics of the wild-type channel. Leucine substitution produced results similar to the T153I mutant channel. This study provides direct electrophysiological evidence for the structure and function of the Kir7.1 channel's narrow inner pore in regulating conductance.

Investigating the Mechanistic Strategies of the Two‐component FMN‐dependent Alkanesulfonate Monooxygenase Systems

The two‐component alkanesulfonate monooxygenase systems, consisting of a flavin reductase (SsuE and MsuE) and an alkanesulfonate monooxygenase (SsuD and MsuD), enable bacterial organisms to utilize a broad range of alkanesulfonates when sulfur is limiting. The flavin reductases supply reduced flavin to their partner monooxygenase for the desulfonation of sulfonated compounds through the formation of a flavin oxygenating intermediate. Commonly, flavin monooxygenases have been proposed to utilize a C4a‐(hydro)peroxyflavin as the oxygenating intermediate. However, unlike other flavin monooxygenase enzymes, this intermediate has not been spectrally observed in the alkanesulfonate monooxygenase enzymes. Recent studies reported the formation of a flavin‐N5‐oxide which forms as an intermediate during turnover or as the final product in some flavin monooxygenases. It is hypothesized that the alkanesulfonate monooxygenases could employ a flavin‐N5‐adduct based on amino acid sequence alignments and structural similarities with enzymes employing similar intermediates.SsuD and MsuD share comparable structural properties but have different specificities for alkanesulfonate substrates. Although the substrate preference is critical for catalytic competence, SsuD and MsuD likely utilize similar catalytic steps for desulfonation. Previous studies identified various polar and nonpolar amino acid residues that were critical for the formation and stabilization of the flavin‐N5‐oxide. The conserved nonpolar amino acids are proposed to control the interaction of the flavin‐N5 with molecular oxygen, whereas the polar amino acids are involved in stabilizing the superoxide anion involved in the formation of the flavin‐N5 oxygenating intermediate. Some of these amino acids were also found to be conserved in the alkanesulfonate monooxygenases and their role was further evaluated through site‐directed mutagenesis. The V108T and T109A SsuD variants had similar activity to wild‐type SsuD; however, the N108L SsuD variant had no measurable activity. These results support the role of Asn in stabilizing the proposed flavin‐N5 oxygenating intermediate. Since a flavin‐N5‐oxide has been identified as an intermediate or as the final product in some two‐component monooxygenases, HPLC and mass spectrometric analyses were performed to determine if the alkanesulfonate monooxygenases form the flavin‐N5‐oxide. To provide evidence for kinetic steps and identify flavin‐N5 reaction intermediates, stopped‐flow kinetic experiments were performed with wild‐type and variants of SsuD and MsuD. The findings from this study provide insight into the mechanistic features of the alkanesulfonate monooxygenases as well as their role in the overall global sulfur cycle.

Modeling Dehalogenase PceA to Evaluate Pollutant Organohalide Biodegradation

Per‐ and polyfluoroalkyl substances (PFAS) are ubiquitous pollutants found in water and land environments. Environmental buildup of organohalides, such as perchloroethylene (PCE) and trichloroethene (TCE), used in dry cleaning, firefighting foams, and food packaging, pose an increasing public health risk due to their slow degradation. PFAS molecules are now observed in the blood of humans and animals across the world, and their full impact on health is yet to be determined. Studies of PFAS biodegradation have been reported within various microbial organisms. One reductive dehalogenase identified is PceA from Sulfurospirillium multivorans. The Summit Country Day School MSOE Center for Biomolecular Modeling MAPS team used 3D modeling and printing technology to investigate the active site of the reductive dehalogenase PceA. PceA is a homodimer with two independent active sites. Each active site contains a norpseudo‐B12, two 4Fe‐4S clusters as well as three highly conserved amino acids, Tyr246, Arg305, and Asn272. The short distances between the proximal iron‐sulfur cluster and the cobalt ion bound to the corrin ring of norpseudo‐B12 are thought to allow rapid electron transfer for catalysis. The invariant Tyr246 can donate a proton to neutralize the carbanion formed during catalysis. Deprotonated Tyr246 may be stabilized by the guanidinium side chain of Arg305. Additionally, the active sites have a hydrophobic pocket lined with bulky nonpolar amino acids Phe38, Trp56, Tyr102, Trp96, Tyr382 and Trp376. The tightly packed hydrophobic sidechains provide a gate for substrate selection. Understanding PFAS biodegradation and identifying additional reductive dehalogenases is urgent in addressing the pollution that faces our environment and critical in ensuring the health of humans and ecosystems.

The Escherichia coli Amino Acid Uptake Protein CycA: Regulation of Its Synthesis and Practical Application in l-Isoleucine Production.

Amino acid transport systems perform important physiological functions; their role should certainly be considered in microbial production of amino acids. Typically, in the context of metabolic engineering, efforts are focused on the search for and application of specific amino acid efflux pumps. However, in addition, importers can also be used to improve the industrial process as a whole. In this study, the protein CycA, which is known for uptake of nonpolar amino acids, was characterized from the viewpoint of regulating its expression and range of substrates. We prepared a cycA-overexpressing strain and found that it exhibited high sensitivity to branched-chain amino acids and their structural analogues, with relatively increased consumption of these amino acids, suggesting that they are imported by CycA. The expression of cycA was found to be dependent on the extracellular concentrations of substrate amino acids. The role of some transcription factors in cycA expression, including of Lrp and Crp, was studied using a reporter gene construct. Evidence for the direct binding of Crp to the cycA regulatory region was obtained using a gel-retardation assay. The enhanced import of named amino acids due to cycA overexpression in the l-isoleucine-producing strain resulted in a significant reduction in the generation of undesirable impurities. This work demonstrates the importance of uptake systems with respect to their application in metabolic engineering.