bacterial enzyme
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AbstractAt the joint between the membrane and hydrophilic arms of the enzyme, the structure of the respiratory complex I reveals a tunnel-like Q-chamber for ubiquinone binding and reduction. The narrow entrance of the quinone chamber located in ND1 subunit forms a bottleneck (eye of a needle) which in all resolved structures was shown to be too small for a bulky quinone to pass through, and it was suggested that a conformational change is required to open the channel. The closed bottleneck appears to be a well-established feature of all structures reported so-far, both for the so-called open and closed states of the enzyme, with no indication of a stable open state of the bottleneck. We propose a squeeze-in mechanism of the bottleneck passage, where dynamic thermal conformational fluctuations allow quinone to get in and out. Here, using molecular dynamics simulations of the bacterial enzyme, we have identified collective conformational changes that open the quinone chamber bottleneck. The model predicts a significant reduction—due to a need for a rare opening of the bottleneck—of the effective bi-molecular rate constant, in line with the available kinetic data. We discuss possible reasons for such a tight control of the quinone passage into the binding chamber and mechanistic consequences for the quinone two-electron reduction. Graphic abstract

Aims: This study attempted to evaluate the five host strains, including BL21 (DE3), Rosetta (DE3), DH5α, XL1-BLUE, and SHuffle, in terms of arginine deiminase (ADI) production and enzyme activity. Background: Escherichia coli is one of the most preferred host microorganisms for the production of recombinant proteins due to its well-characterized genome, availability of various expression vectors, and host strains. Choosing a proper host strain for the overproduction of a desired recombinant protein is very important because of the diversity of genetically modified expression strains. Various E. coli cells have been examined in different patent applications. Method: ADI was chosen as a bacterial enzyme that degrades L-arginine. It is effective in the treatment of some types of human cancers like melanoma and hepatocellular carcinoma (HCC), which are arginine-auxotrophic. Five mentioned E. coli strains were cultivated. The pET-3a was used as the expression vector. The competent E. coli cells were obtained through the CaCl2 method. It was then transformed with the construct of pET3a-ADI using the heat shock strategy. The ADI production levels were examined by 10% SDS-PAGE analysis. The ability of host strains for the expression of the requested recombinant protein was compared. The enzymatic activity of the obtained recombinant ADI from each studied strain was assessed by a colorimetric 96-well microtiter plate assay. Result: All the five strains exhibited a significant band at 46 kDa. BL21 (DE3) produced the highest amount of ADI protein, followed by Rosetta (DE3). The following activity assay showed that ADI from BL21 (DE3) and Rosetta (DE3) had the most activity. Conclusion: There are some genetic and metabolic differences among the various E. coli strains, leading to differences in the amount of recombinant protein production. The results of this study can be used for the efficacy evaluation of the five studied strains for the production of similar pharmaceutical enzymes. The strains also could be analyzed in terms of proteomics.

The ubiquitous persistence of plastic waste in diverse forms and different environmental matrices is one of the main challenges that modern societies are facing at present. The exponential utilization and recalcitrance of synthetic plastics, including polyethylene terephthalate (PET), results in their extensive accumulation, which is a significant threat to the ecosystem. The growing amount of plastic waste ending up in landfills and oceans is alarming due to its possible adverse effects on biota. Thus, there is an urgent need to mitigate plastic waste to tackle the environmental crisis of plastic pollution. With regards to PET, there is a plethora of literature on the transportation route, ingestion, environmental fate, amount, and the adverse ecological and human health effects. Several studies have described the deployment of various microbial enzymes with much focus on bacterial-enzyme mediated removal and remediation of PET. However, there is a lack of consolidated studies on the exploitation of fungal enzymes for PET degradation. Herein, an effort has been made to cover this literature gap by spotlighting the fungi and their unique enzymes, e.g., esterases, lipases, and cutinases. These fungal enzymes have emerged as candidates for the development of biocatalytic PET degradation processes. The first half of this review is focused on fungal biocatalysts involved in the degradation of PET. The latter half explains three main aspects: (1) catalytic mechanism of PET hydrolysis in the presence of cutinases as a model fungal enzyme, (2) limitations hindering enzymatic PET biodegradation, and (3) strategies for enhancement of enzymatic PET biodegradation.

Loss of metabolic homeostasis is a hallmark of aging and is characterized by dramatic metabolic reprogramming. To analyze how the fate of labeled methionine is altered during aging, we applied 13C5-Methionine labeling to Drosophila and demonstrated significant changes in the activity of different branches of the methionine metabolism as flies age. We further tested whether targeted degradation of methionine metabolism components would “reset” methionine metabolism flux and extend the fly lifespan. Specifically, we created transgenic flies with inducible expression of Methioninase, a bacterial enzyme capable of degrading methionine and revealed methionine requirements for normal maintenance of lifespan. We also demonstrated that microbiota-derived methionine is an alternative and important source in addition to food-derived methionine. In this genetic model of methionine restriction (MetR), we also demonstrate that either whole-body or tissue-specific Methioninase expression can dramatically extend Drosophila health- and lifespan and exerts physiological effects associated with MetR. Interestingly, while previous dietary MetR extended lifespan in flies only in low amino acid conditions, MetR from Methioninase expression extends lifespan independently of amino acid levels in the food. Finally, because impairment of the methionine metabolism has been previously associated with the development of Alzheimer’s disease, we compared methionine metabolism reprogramming between aging flies and a Drosophila model relevant to Alzheimer’s disease, and found that overexpression of human Tau caused methionine metabolism flux reprogramming similar to the changes found in aged flies. Altogether, our study highlights Methioninase as a potential agent for health- and lifespan extension.

SARM1 is an inducible NAD+ hydrolase that is the central executioner of pathological axon loss. Recently, we elucidated the molecular mechanism of SARM1 activation, demonstrating that SARM1 is a metabolic sensor regulated by the levels of NAD+ and its precursor, nicotinamide mononucleotide (NMN), via their competitive binding to an allosteric site. In healthy neurons with abundant NAD+, binding of NAD+ blocks access of NMN to this allosteric site. However, with injury or disease the levels of the NAD+ biosynthetic enzyme NMNAT2 drop, increasing the NMN/NAD+ ratio and thereby promoting NMN binding to the SARM1 allosteric site, which in turn induces a conformational change activating the SARM1 NAD+ hydrolase. Hence, NAD+ metabolites both regulate the activation of SARM1 and, in turn, are regulated by the SARM1 NAD+ hydrolase. This dual upstream and downstream role for NAD+ metabolites in SARM1 function has hindered mechanistic understanding of axoprotective mechanisms that manipulate the NAD+ metabolome. Here we reevaluate two methods that potently block axon degeneration via modulation of NAD+ related metabolites, 1) the administration of the NMN biosynthesis inhibitor FK866 in conjunction with the NAD+ precursor nicotinic acid riboside (NaR) and 2) the neuronal expression of the bacterial enzyme NMN deamidase. We find that these approaches not only lead to a decrease in the levels of the SARM1 activator NMN, but also an increase in the levels of the NAD+ precursor nicotinic acid mononucleotide (NaMN). We show that NaMN competes with NMN for binding to the SARM1 allosteric site, that NaMN inhibits SARM1 activation, and that this NaMN-mediated inhibition is important for the long-term axon protection induced by these treatments. Together, these results demonstrate that the SARM1 allosteric pocket can bind a diverse set of metabolites including NMN, NAD+, and NaMN to monitor cellular NAD+ homeostasis and regulate SARM1 NAD+ hydrolase activity. The relative promiscuity of the allosteric site may enable the development of potent pharmacological inhibitors of SARM1 activation for the treatment of neurodegenerative disorders.

The study is aimed to develop a relevant synbiotic to promote growth performance of the whiteleg shrimp, Litopenaeus vannamei. For this, four common natural fiber extracts from Arcera banana, Siamese banana, yellow sweet potato, and white sweet potato were screened for supporting the growth of Lactobacillus sp. which was isolated from whiteleg shrimp intestines with probiotic activity, prebiotic score, and ability to induce bacterial enzyme activities of protease, leu-aminopeptidase, and a-amylase. Results showed that Lactobacillus sp. was able to utilize all extracts from banana and sweet potato as the sole carbon sources. At 24 hours of culture, the growth of Lactobacillus sp. was highest after adding the extract from white sweet potato as the sole carbon source. Considering pathogenic bacteria, including Vibrio parahaemolyticus, white sweet potato extract had the highest prebiotic score with a mean of 0.25 as compared with those of V. harveyi with a mean of 0.16. White sweet potato extract induced the highest activities of protease. These results indicated that white sweet potato extract was more suitable for combining with Lactobacillus sp. as a synbiotic for shrimp culture.

Solubility is a requirement for many cellular processes. Loss of solubility and aggregation can lead to the partial or complete abrogation of protein function. Thus, understanding the relationship between protein evolution and aggregation is an important goal. Here, we analysed two deep mutational scanning experiments to investigate the role of protein aggregation in molecular evolution. In one data set, mutants of a protein involved in RNA biogenesis and processing, human TAR DNA binding protein 43 (TDP-43), were expressed in S. cerevisiae. In the other data set, mutants of a bacterial enzyme that controls resistance to penicillins and cephalosporins, TEM-1 beta-lactamase, were expressed in E. coli under the selective pressure of an antibiotic treatment. We found that aggregation differentiates the effects of mutations in the two different cellular contexts. Specifically, aggregation was found to be associated with increased cell fitness in the case of TDP-43 mutations, as it protects the host from aberrant interactions. By contrast, in the case of TEM-1 beta-lactamase mutations, aggregation is linked to a decreased cell fitness due to inactivation of protein function. Our study shows that aggregation is an important context-dependent constraint of molecular evolution and opens up new avenues to investigate the role of aggregation in the cell.

Viruses are ubiquitous pathogens of global impact. Prompted by the hypothesis that their earliest progenitors recruited host proteins for virion formation, we have used stringent laboratory evolution to convert a bacterial enzyme that lacks affinity for nucleic acids into an artificial nucleocapsid that efficiently packages and protects multiple copies of its own encoding messenger RNA. Revealing remarkable convergence on the molecular hallmarks of natural viruses, the accompanying changes reorganized the protein building blocks into an interlaced 240-subunit icosahedral capsid that is impermeable to nucleases, and emergence of a robust RNA stem-loop packaging cassette ensured high encapsidation yields and specificity. In addition to evincing a plausible evolutionary pathway for primordial viruses, these findings highlight practical strategies for developing nonviral carriers for diverse vaccine and delivery applications.