Allosteric regulation of protein function is a mechanism by which an event in one place of a protein structure causes an effect at another site, much like the behavior of a telecommunications network ...in which a collection of transmitters, receivers and transceivers communicate with each other across long distances. For example, ligand binding or an amino acid mutation at an allosteric site can alter enzymatic activity or binding affinity in a distal region such as the active site or a second binding site. The mechanism of this site-to-site communication is of great interest, especially since allosteric effects must be considered in drug design and protein engineering. In this review, conformational mobility as the common route between allosteric regulation and catalysis is discussed. We summarize recent experimental data and the resulting insights into allostery within proteins, and we discuss the nature of future studies and the new applications that may result from increased understanding of this regulatory mechanism.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
Lymphatic filariasis is a debilitating illness with an estimated 50 million cases as of 2018. The majority of cases are caused by the parasitic worm W. bancrofti and additional cases by the worms B. ...malayi and B. timori. Dihydrofolate reductase (DHFR) is an established target in the treatment of cancer, bacterial, and protozoal infections and may be a potential target for drugs targeting parasitic worm infections, including filariasis. Recent studies have shown that known antifolate compounds, including methotrexate, inhibit the activity of W. bancrofti DHFR (WbDHFR). However, the absence of structural information for filarial DHFRs has limited the study of more in-depth structure-function relationships. We report the structure of WbDHFR complexed with NADPH and folate using X-ray diffraction data measured to 2.47 Å resolution. The structure of WbDHFR reveals the usual DHFR fold and is currently only the second nematode DHFR structure in the Protein Data Bank. The equilibrium dissociation constants for NADPH (90 ± 29 nM) and folate (23 ± 4 nM) were determined by equilibrium titrations. The interactions of known antifolates with WbDHFR were analyzed using molecular docking programs and molecular dynamics simulations. Antifolates with a hydrophobic core and extended linker formed favorable interactions with WbDHFR. These combined data should now facilitate the rational design of filarial DHFR inhibitors, which in turn can be used to determine whether DHFR is a viable drug target for filariasis and whether existing antifolates may be repurposed for its treatment.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
The ubiquity of urban brownfields presents not only a challenge for environmental managers but also an opportunity to study the functional aspects of degraded ecosystems that are in close contact ...with human habitation. In this study, we investigate the soil microbial community response to heavy metal contamination at Liberty State Park (LSP), an urban brownfield in Jersey City, NJ, USA. Heavy metal contamination of the soils at LSP is heterogeneous, varying widely across site and among metals. We collected soils along a previously mapped gradient of metal contamination at LSP and sampled soil from a local and uncontaminated reference site (Hutcheson Memorial Forest (HMF)) for comparison. For all soils, we measured soil heavy metal concentrations, soil organic carbon content, bacterial density, and extracellular phosphatase activity as a proxy of ecosystem functioning. Additionally, we analyzed the microbial community composition using high-throughput sequencing. Data show that some sites within LSP have significantly higher phosphatase activity compared to HMF, indicating that some heavily contaminated LSP soils are highly functional. We also found that soil organic carbon and bacterial density have a significant and positive relationship with phosphatase activity. The microbial community analyses showed that the bacterial communities were sensitive to heavy metals and that the composition was significantly affected in particular by copper, zinc, and lead. The fungal communities, however, did not vary significantly with heavy metals. Our results shed important light on the composition and functioning of urban brownfield soils. A deeper understanding of these unique ecosystems is required for successful remediation, restoration and urban sustainability.
•Bacterial community composition varies with soil metal load, but fungal does not.•Soil microbial community functioning can be high in spite of metal load.•Soil phosphatase activity correlate with factors like: pH, soil organic matter, and bacterial abundance.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
The legacy of industrialization has left many soils contaminated. However, soil organisms and plant communities can thrive in spite of metal contamination and, in some cases, metabolize and help in ...remediation. The responses of plants and soil organisms to contamination are mutually dependent and dynamic. Plant–soil feedbacks are central to the development of any terrestrial community; they are ongoing in both contaminated and healthy soils. However, the theory that governs plant–soil feedbacks in healthy soils needs to be studied in contaminated soils. In healthy soils, negative feedbacks (i.e. pathogens) play a central role in shaping plant community structure. However to our knowledge, the nature of feedback relationships has never been addressed in contaminated soils. Here we review literature that supports a plant–soil feedback approach to understanding the ecology of metal-contaminated soil. Further, we discuss the idea that within these soils, the role of positive as opposed to negative plant–soil feedbacks may be more important. Testing this idea in a rigorous way in any ecosystem is challenging, and metal contamination imposes an additional abiotic constraint. We discuss research goals and experimental approaches to study plant–soil interactions applicable to metal-contaminated soils; these insights can be extended to other contaminated environments and restoration efforts.
•Plants, soil organisms and metal contamination interact in dynamic ways.•Plant–soil feedback theory may be subject to challenge under contamination.•Generalized feedback theory in urban soils requires reference sites and meta-analysis.•We have technological tools to study feedback interactions in contaminated soils.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
Protein motions play a fundamental role in enzyme catalysis and ligand binding. The relationship between protein motion and function has been extensively investigated in the model enzyme ...dihydrofolate reductase (DHFR). DHFR is an essential enzyme that catalyzes the reduction of dihydrofolate to tetrahydrofolate. Numerous experimental and computational methods have been used to probe the motions of DHFR through the catalytic cycle and to investigate the effect of distal mutations on DHFR motions and ligand binding. These experimental investigations have pushed forward the study of protein motions and their role in protein-ligand interactions. The introduction of mutations distal to the active site has been shown to have profound effects on ligand binding, hydride transfer rates and catalytic efficacy and these changes are captured by enzyme kinetics measurements. Distal mutations have been shown to exert their effects through a network of correlated amino acids and these effects have been investigated by NMR, protein dynamics, and analysis of coupled amino acids. The experimental methods and the findings that are reviewed here have broad implications for our understanding of enzyme mechanisms, ligand binding and for the future design and discovery of enzyme inhibitors.
Tuberculosis (TB), caused by the pathogen Mycobacterium tuberculosis, affects millions of people worldwide. Several TB drugs have lost efficacy due to emerging drug resistance and new anti‐TB targets ...are needed. Recent research suggests that indole‐3‐glycerol phosphate synthase (IGPS) in M. tuberculosis (MtIGPS) could be such a target. IGPS is a (β/α)8‐barrel enzyme that catalyzes the conversion of 1‐(o‐carboxyphenylamino)‐1‐deoxyribulose 5’‐phosphate (CdRP) into indole‐glycerol‐phosphate (IGP) in the bacterial tryptophan biosynthetic pathway. M. tuberculosis over expresses the tryptophan pathway genes during an immune response and inhibition of MtIGPS allows CD4 T‐cells to more effectively fight against M. tuberculosis. Here we review the published data on MtIGPS expression, kinetics, mechanism, and inhibition. We also discuss MtIGPS crystal structures and compare them to other IGPS structures to reveal potential structure‐function relationships of interest for the purposes of drug design and biocatalyst engineering.
Indole‐3‐glycerol phosphate synthase (IGPS) catalyzes the indole‐forming step in bacterial tryptophan biosynthesis. This minireview summarizes the data on expression, kinetics, mechanism, and inhibition of IGPS from Mycobacterium tuberculosis. Crystal structures are also analyzed and compared to reveal structure‐function relationships of value for the purposes of drug design and biocatalyst engineering.
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FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SAZU, SBCE, SBMB, UL, UM, UPUK
Restoring enzyme function in barren, brownfield soils using green strategies can improve microbial functioning and enable phytoremediation. It is known that adding simple, readily metabolized ...substrates secreted by growing plant roots (root exudates) or a laboratory prepared solution of root exudates (artificial root exudates) can stimulate soil microbial function. It is not known whether and how well this strategy works in a contaminated, low functioning soil from an industrial barren site because contaminants in the barren soil might inhibit microbial survival and functioning, or the microbial community might not be adapted to functionally benefit from root exudates. The objective of this study was to determine whether artificial root exudates stimulate microbial function in a barren soil. We collected soils from a barren brownfield (25R) site and an adjacent vegetated brownfield site (25F), with low and high enzyme activities, respectively. We subjected both soils to three treatments: switchgrass (native to the site), artificial root exudates, and a combination of switchgrass and artificial root exudates. We measured enzymatic activity, plant growth, soil moisture, organic matter content, and easily extractable glomalin content over 205 days. By day 157, artificial root exudates increased the phosphatase activity by 9-fold in previously vegetated brownfield soil and by 351-fold in barren brownfield soil. When exudates were added to the barren soil, the plant shoot mass was higher (52.2 ± 2.5 mg) than when they were not (35.4 ± 3.6 mg). In both soils, adding artificial root exudates significantly increased the percent moisture, organic matter, and glomalin content. Treating contaminated, barren soil with artificial root exudates resulted in increased soil microbial function and improved soil properties that might promote a hospitable habitat to support vegetation in such extreme environments.
Summary: We added artificial root exudates to stimulate enzymatic function in two contaminated soils. Plant shoot mass, soil percent moisture, glomalin content, and organic matter content significantly increased due to the addition of artificial root exudates to the study soils. Microbially-mediated phosphatase activity was established in a barren, previously inactive, polluted soil.
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•A barren, contaminated, and enzymatically inactive industrial soil was revitalized.•Artificial root exudates increased the barren soil's phosphatase activity >300-fold.•Activity increased 39-fold more in the barren soil than in a vegetated reference.•Plant biomass and glomalin content also significantly increased in the barren soil.•After treatment, the barren soil functionally resembled the vegetated reference soil.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Filariasis is a tropical disease caused by the parasitic nematodes Wuchereria bancrofti and Brugia malayi. Known inhibitors of dihydrofolate reductase (DHFR) have been previously shown to kill Brugia ...malayi nematodes and to inhibit Brugia malayi DHFR (BmDHFR) at nanomolar concentrations. These data suggest that BmDHFR is a potential target for the treatment of filariasis. Here, protocols for cloning, expression and purification of Wuchereria bancrofti DHFR (WbDHFR) were developed. The Uniprot entry J9F199-1 predicts a 172 amino acid protein for WbDHFR but alignment of this sequence to the previously described BmDHFR shows that this WbDHFR sequence lacks a crucial, conserved 13 amino acid loop. The presence of the loop in WbDHFR is supported by a noncanonical splicing event and the loop sequence was therefore included in the gene design. Subsequently, the KM for dihydrofolate (3.7 ± 2 μM), kcat (7.4 ± 0.6 s-1), and pH dependence of activity were determined. IC50 values of methotrexate, trimethoprim, pyrimethamine, raltitrexed, aminopterin, (-)-epicatechin gallate, (-)-epicatechin, and vitexin were measured for WbDHFR and BmDHFR. Methotrexate and structurally related aminopterin were found to be effective inhibitors of WbDHFR, with an KI of 1.2 ± 0.2 nM and 2.1 ± 0.5 nM, respectively, suggesting that repurposing of known antifolate compound may be an effective strategy to treating filariasis. Most compounds showed similar inhibition profiles toward both enzymes, suggesting that the two enzymes have important similarities in their active site environments and can be targeted with the same compound, once a successful inhibitor is identified.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
One of the most intriguing questions in modern enzymology is whether enzyme dynamics evolved to enhance the catalyzed chemical transformation. In this study, dihydrofolate reductase, a small ...monomeric protein that catalyzes a single C-H-C transfer, is used as a model system to address this question. Experimental and computational studies have proposed a dynamic network that includes two residues remote from the active site (G121 and M42). The current study compares the nature of the H-transfer step of the WT enzyme, two single mutants, and their double mutant. The contribution of quantum mechanical tunneling and enzyme dynamics to the H-transfer step was examined by determining intrinsic kinetic isotope effects, their temperature dependence, and activation parameters. Different patterns of environmentally coupled tunneling were found for these four enzymes. The findings indicate that the naturally evolved WT dihydrofolate reductase requires no donor-acceptor distance fluctuations (no gating). Both single mutations affect the rearrangement of the system before tunneling, so some gating is required, but the overall nature of the environmentally coupled tunneling appears similar to that of the WT enzyme. The double mutation, on the other hand, seems to cause a major change in the nature of H transfer, leading to poor reorganization and substantial gating. These findings support the suggestion that these distal residues synergistically affect the H transfer at the active site of the enzyme. This observation is in accordance with the notion that these remote residues are part of a dynamic network that is coupled to the catalyzed chemistry.
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BFBNIB, NMLJ, NUK, PNG, SAZU, UL, UM, UPUK
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