2023

Jump to: 2022, 2021

20 pages, 2138 KiB

Open AccessArticle

Two-Photon Excited Fluorescence of NADH-Alcohol Dehydrogenase Complex in a Mixture with Bacterial Enzymes

by Ioanna A. Gorbunova, Maxim E. Sasin, Dmitry V. Yachkov, Denis A. Volkov, Alexei D. Vedyaykin, Andrey A. Nikiforov and Oleg S. Vasyutinskii

Biomolecules 2023, 13(2), 256; https://doi.org/10.3390/biom13020256 - 30 Jan 2023

Cited by 3 | Viewed by 1636

Abstract

Thorough study of composition and fluorescence properties of a commercial reagent of active equine NAD-dependent alcohol dehydrogenase expressed and purified from E. coli has been carried out. Several experimental methods: spectral- and time-resolved two-photon excited fluorescence, sodium dodecyl sulfate–polyacrylamide gel electrophoresis, fast protein [...] Read more.

Thorough study of composition and fluorescence properties of a commercial reagent of active equine NAD-dependent alcohol dehydrogenase expressed and purified from E. coli has been carried out. Several experimental methods: spectral- and time-resolved two-photon excited fluorescence, sodium dodecyl sulfate–polyacrylamide gel electrophoresis, fast protein liquid chromatography, and mass spectrometry were used for analysis. The reagent under study was found to contain also a number of natural fluorophores: free NAD(P)H, NADH-alcohol dehydrogenase, NADPH-isocitrate dehydrogenase, and pyridoxal 5-phosphate—serine hydroxymethyltransferase complexes. The results obtained demonstrated the potential and limitations of popular optical methods as FLIM for separation of fluorescence signals from free and protein-bound forms of NADH, NADPH, and FAD that are essential coenzymes in redox reactions in all living cells. In particular, NADH-alcohol dehydrogenase and NADPH-isocitrate dehydrogenase complexes could not be optically separated in our experimental conditions although fast protein liquid chromatography and mass spectrometry analysis undoubtedly indicated the presence of both enzymes in the molecular sample used. Also, the results of fluorescence, fast protein liquid chromatography, and mass spectrometry analysis revealed a significant contribution of the enzyme-bound coenzyme pyridoxal 5-phosphate to the fluorescence signal that could be separated from enzyme-bound NADH by using bandpass filters, but could effectively mask contribution from enzyme-bound FAD because the fluorescence spectra of the species practically overlapped. It was shown that enzyme-bound pyridoxal 5-phosphate fluorescence can be separated from enzyme-bound NAD(P)H and FAD through analysis of short fluorescence decay times of about tens of picoseconds. However, this analysis was found to be effective only at relatively high number of peak photon counts in recorded fluorescence signals. The results obtained in this study can be used for interpretation of fluorescence signals from a mixture of enzyme-bound fluorophores and should be taken into consideration when determining the intracellular NADH/FAD ratio using FLIM. Full article

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2022

Jump to: 2023, 2021

20 pages, 3325 KiB

Open AccessEditor’s ChoiceArticle

Structural Insights into the Dimeric Form of Bacillus subtilis RNase Y Using NMR and AlphaFold

by Nelly Morellet, Pierre Hardouin, Nadine Assrir, Carine van Heijenoort and Béatrice Golinelli-Pimpaneau

Biomolecules 2022, 12(12), 1798; https://doi.org/10.3390/biom12121798 - 01 Dec 2022

Cited by 3 | Viewed by 1981

Abstract

RNase Y is a crucial component of genetic translation, acting as the key enzyme initiating mRNA decay in many Gram-positive bacteria. The N-terminal domain of Bacillus subtilis RNase Y (Nter-BsRNaseY) is thought to interact with various protein partners within a degradosome complex. Bioinformatics [...] Read more.

RNase Y is a crucial component of genetic translation, acting as the key enzyme initiating mRNA decay in many Gram-positive bacteria. The N-terminal domain of Bacillus subtilis RNase Y (Nter-BsRNaseY) is thought to interact with various protein partners within a degradosome complex. Bioinformatics and biophysical analysis have previously shown that Nter-BsRNaseY, which is in equilibrium between a monomeric and a dimeric form, displays an elongated fold with a high content of α-helices. Using multidimensional heteronuclear NMR and AlphaFold models, here, we show that the Nter-BsRNaseY dimer is constituted of a long N-terminal parallel coiled-coil structure, linked by a turn to a C-terminal region composed of helices that display either a straight or bent conformation. The structural organization of the N-terminal domain is maintained within the AlphaFold model of the full-length RNase Y, with the turn allowing flexibility between the N- and C-terminal domains. The catalytic domain is globular, with two helices linking the KH and HD modules, followed by the C-terminal region. This latter region, with no function assigned up to now, is most likely involved in the dimerization of B. subtilis RNase Y together with the N-terminal coiled-coil structure. Full article

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12 pages, 1696 KiB

Open AccessArticle

Identification, Characterization, and Expression of a β-Galactosidase from Arion Species (Mollusca)

by Julia Thoma, David Stenitzer, Reingard Grabherr and Erika Staudacher

Biomolecules 2022, 12(11), 1578; https://doi.org/10.3390/biom12111578 - 27 Oct 2022

Cited by 4 | Viewed by 1628

Abstract

β-Galactosidases (β-Gal, EC 3.2.1.23) catalyze the cleavage of terminal non-reducing β-D-galactose residues or transglycosylation reactions yielding galacto-oligosaccharides. In this study, we present the isolation and characterization of a β-galactosidase from Arion lusitanicus, and based on this, the cloning and expression of a [...] Read more.

β-Galactosidases (β-Gal, EC 3.2.1.23) catalyze the cleavage of terminal non-reducing β-D-galactose residues or transglycosylation reactions yielding galacto-oligosaccharides. In this study, we present the isolation and characterization of a β-galactosidase from Arion lusitanicus, and based on this, the cloning and expression of a putative β-galactosidase from Arion vulgaris (A0A0B7AQJ9) in Sf9 cells. The entire gene codes for a protein consisting of 661 amino acids, comprising a putative signal peptide and an active domain. Specificity studies show exo- and endo-cleavage activity for galactose β1,4-linkages. Both enzymes, the recombinant from A. vulgaris and the native from A. lusitanicus, display similar biochemical parameters. Both β-galactosidases are most active in acidic environments ranging from pH 3.5 to 4.5, and do not depend on metal ions. The ideal reaction temperature is 50 °C. Long-term storage is possible up to +4 °C for the A. vulgaris enzyme, and up to +20 °C for the A. lusitanicus enzyme. This is the first report of the expression and characterization of a mollusk exoglycosidase. Full article

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2021

Jump to: 2023, 2022

13 pages, 831 KiB

Open AccessReview

Regulation of the Immune Checkpoint Indoleamine 2,3-Dioxygenase Expression by Epstein–Barr Virus

by Leila Sawada, Antonio Carlos Rosário Vallinoto and Igor Brasil-Costa

Biomolecules 2021, 11(12), 1792; https://doi.org/10.3390/biom11121792 - 30 Nov 2021

Cited by 5 | Viewed by 2044

Abstract

Epstein–Barr virus (EBV) is an oncovirus ubiquitously distributed and associated with different types of cancer. The reason why only a group of infected people develop cancer is still unknown. EBV-associated cancers represent about 1.8% of all cancer deaths worldwide, with more than 150,000 [...] Read more.

Epstein–Barr virus (EBV) is an oncovirus ubiquitously distributed and associated with different types of cancer. The reason why only a group of infected people develop cancer is still unknown. EBV-associated cancers represent about 1.8% of all cancer deaths worldwide, with more than 150,000 new cases of cancer being reported annually. Since EBV-associated cancers are described as more aggressive and more resistant to the usual treatment compared to EBV-negative ones, the recent introduction of monoclonal antibodies (mAbs) targeting immune checkpoints (ICs) in the treatment of cancer patients represents a possible therapy for EBV-associated diseases. However, the current mAb therapies available still need improvement, since a group of patients do not respond well to treatment. Therefore, the main objective of this review is to summarize the progress made regarding the contribution of EBV infection to the expression of the IC indoleamine 2,3-dioxygenase (IDO) thus far. This IC has the potential to be used as a target in new immune therapies, such as mAbs. We hope that this work helps the development of future immunotherapies, improving the prognosis of EBV-associated cancer patients. Full article

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15 pages, 2861 KiB

Open AccessArticle

Novel Bi-Modular GH19 Chitinase with Broad pH Stability from a Fibrolytic Intestinal Symbiont of Eisenia fetida, Cellulosimicrobium funkei HY-13

by Lu Bai, Jonghoon Kim, Kwang-Hee Son, Chung-Wook Chung, Dong-Ha Shin, Bon-Hwan Ku, Do Young Kim and Ho-Yong Park

Biomolecules 2021, 11(11), 1735; https://doi.org/10.3390/biom11111735 - 21 Nov 2021

Cited by 6 | Viewed by 1971

Abstract

Endo-type chitinase is the principal enzyme involved in the breakdown of N-acetyl-d-glucosamine-based oligomeric and polymeric materials through hydrolysis. The gene (966-bp) encoding a novel endo-type chitinase (ChiJ), which is comprised of an N-terminal chitin-binding domain type 3 and a C-terminal [...] Read more.

Endo-type chitinase is the principal enzyme involved in the breakdown of N-acetyl-d-glucosamine-based oligomeric and polymeric materials through hydrolysis. The gene (966-bp) encoding a novel endo-type chitinase (ChiJ), which is comprised of an N-terminal chitin-binding domain type 3 and a C-terminal catalytic glycoside hydrolase family 19 domain, was identified from a fibrolytic intestinal symbiont of the earthworm Eisenia fetida, Cellulosimicrobium funkei HY-13. The highest endochitinase activity of the recombinant enzyme (rChiJ: 30.0 kDa) toward colloidal shrimp shell chitin was found at pH 5.5 and 55 °C and was considerably stable in a wide pH range (3.5–11.0). The enzyme exhibited the highest biocatalytic activity (338.8 U/mg) toward ethylene glycol chitin, preferentially degrading chitin polymers in the following order: ethylene glycol chitin > colloidal shrimp shell chitin > colloidal crab shell chitin. The enzymatic hydrolysis of N-acetyl-β-d-chitooligosaccharides with a degree of polymerization from two to six and colloidal shrimp shell chitin yielded primarily N,N′-diacetyl-β-d-chitobiose together with a small amount of N-acetyl-d-glucosamine. The high chitin-degrading ability of inverting rChiJ with broad pH stability suggests that it can be exploited as a suitable biocatalyst for the preparation of N,N′-diacetyl-β-d-chitobiose, which has been shown to alleviate metabolic dysfunction associated with type 2 diabetes. Full article

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22 pages, 2759 KiB

Open AccessArticle

Kinetic Analysis of the Interaction of Nicking Endonuclease BspD6I with DNA

by Liudmila A. Abrosimova, Nikita A. Kuznetsov, Natalia A. Astafurova, Anastasiia R. Samsonova, Andrey S. Karpov, Tatiana A. Perevyazova, Tatiana S. Oretskaya, Olga S. Fedorova and Elena A. Kubareva

Biomolecules 2021, 11(10), 1420; https://doi.org/10.3390/biom11101420 - 28 Sep 2021

Viewed by 2320

Abstract

Nicking endonucleases (NEs) are enzymes that incise only one strand of the duplex to produce a DNA molecule that is ‘nicked’ rather than cleaved in two. Since these precision tools are used in genetic engineering and genome editing, information about their mechanism of [...] Read more.

Nicking endonucleases (NEs) are enzymes that incise only one strand of the duplex to produce a DNA molecule that is ‘nicked’ rather than cleaved in two. Since these precision tools are used in genetic engineering and genome editing, information about their mechanism of action at all stages of DNA recognition and phosphodiester bond hydrolysis is essential. For the first time, fast kinetics of the Nt.BspD6I interaction with DNA were studied by the stopped-flow technique, and changes of optical characteristics were registered for the enzyme or DNA molecules. The role of divalent metal cations was estimated at all steps of Nt.BspD6I–DNA complex formation. It was demonstrated that divalent metal ions are not required for the formation of a non-specific complex of the protein with DNA. Nt.BspD6I bound five-fold more efficiently to its recognition site in DNA than to a random DNA. DNA bending was confirmed during the specific binding of Nt.BspD6I to a substrate. The optimal size of Nt.BspD6I’s binding site in DNA as determined in this work should be taken into account in methods of detection of nucleic acid sequences and/or even various base modifications by means of NEs. Full article

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25 pages, 4160 KiB

Open AccessEditor’s ChoiceReview

Amylomaltases in Extremophilic Microorganisms

by Claudia Leoni, Bruno A. R. Gattulli, Graziano Pesole, Luigi R. Ceci and Mariateresa Volpicella

Biomolecules 2021, 11(9), 1335; https://doi.org/10.3390/biom11091335 - 09 Sep 2021

Cited by 8 | Viewed by 3082

Abstract

Amylomaltases (4-α-glucanotransferases, E.C. 2.4.1.25) are enzymes which can perform a double-step catalytic process, resulting in a transglycosylation reaction. They hydrolyse glucosidic bonds of α-1,4′-d-glucans and transfer the glucan portion with the newly available anomeric carbon to the 4′-position of an α-1,4′- [...] Read more.

Amylomaltases (4-α-glucanotransferases, E.C. 2.4.1.25) are enzymes which can perform a double-step catalytic process, resulting in a transglycosylation reaction. They hydrolyse glucosidic bonds of α-1,4′-d-glucans and transfer the glucan portion with the newly available anomeric carbon to the 4′-position of an α-1,4′-d-glucan acceptor. The intramolecular reaction produces a cyclic α-1,4′-glucan. Amylomaltases can be found only in prokaryotes, where they are involved in glycogen degradation and maltose metabolism. These enzymes are being studied for possible biotechnological applications, such as the production of (i) sugar substitutes; (ii) cycloamyloses (molecules larger than cyclodextrins), which could potentially be useful as carriers and encapsulating agents for hydrophobic molecules and also as effective protein chaperons; and (iii) thermoreversible starch gels, which could be used as non-animal gelatin substitutes. Extremophilic prokaryotes have been investigated for the identification of amylomaltases to be used in the starch modifying processes, which require high temperatures or extreme conditions. The aim of this article is to present an updated overview of studies on amylomaltases from extremophilic Bacteria and Archaea, including data about their distribution, activity, potential industrial application and structure. Full article

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26 pages, 9941 KiB

Open AccessFeature PaperArticle

The Impact of Lung Proteases on Snake-Derived Antimicrobial Peptides

by Shannice E. Creane, Simon R. Carlile, Damian Downey, Sinéad Weldon, John P. Dalton and Clifford C. Taggart

Biomolecules 2021, 11(8), 1106; https://doi.org/10.3390/biom11081106 - 27 Jul 2021

Cited by 5 | Viewed by 3119

Abstract

Respiratory infections are a leading cause of global morbidity and mortality and are of significant concern for individuals with chronic inflammatory lung diseases. There is an urgent need for novel antimicrobials. Antimicrobial peptides (AMPs) are naturally occurring innate immune response peptides with therapeutic [...] Read more.

Respiratory infections are a leading cause of global morbidity and mortality and are of significant concern for individuals with chronic inflammatory lung diseases. There is an urgent need for novel antimicrobials. Antimicrobial peptides (AMPs) are naturally occurring innate immune response peptides with therapeutic potential. However, therapeutic development has been hindered by issues with stability and cytotoxicity. Availing of direct drug delivery to the affected site, for example the lung, can reduce unwanted systemic side effects and lower the required dose. As cystic fibrosis (CF) and chronic obstructive pulmonary disease (COPD) lungs typically exhibit elevated protease levels, the aim of this study was to assess their impact on snake-derived AMPs. Peptide cleavage was determined using SDS-PAGE and antimicrobial and anti-inflammatory activities of neutrophil elastase (NE)-incubated peptides were assessed using a radial diffusion assay (RDA) and an in vitro LPS-induced inflammation model, respectively. Although the snake-derived AMPs were found to be susceptible to cleavage by lung proteases including NE, several retained their function following NE-incubation. This facilitated the design of novel truncated derivatives that retained functionality following NE incubation. Snake-derived AMPs are tractable candidate treatments for use in environments that feature elevated NE levels, such as the CF airways. Full article

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15 pages, 4267 KiB

Open AccessArticle

Novel Anti-Fungal d-Laminaripentaose-Releasing Endo-β-1,3-glucanase with a RICIN-like Domain from Cellulosimicrobium funkei HY-13

by Lu Bai, Jonghoon Kim, Kwang-Hee Son, Dong-Ha Shin, Bon-Hwan Ku, Do Young Kim and Ho-Yong Park

Biomolecules 2021, 11(8), 1080; https://doi.org/10.3390/biom11081080 - 22 Jul 2021

Cited by 10 | Viewed by 2211

Abstract

Endo-β-1,3-glucanase plays an essential role in the deconstruction of β-1,3-d-glucan polysaccharides through hydrolysis. The gene (1650-bp) encoding a novel, bi-modular glycoside hydrolase family 64 (GH64) endo-β-1,3-glucanase (GluY) with a ricin-type β-trefoil lectin domain (RICIN)-like domain from Cellulosimicrobium funkei HY-13 was identified [...] Read more.

Endo-β-1,3-glucanase plays an essential role in the deconstruction of β-1,3-d-glucan polysaccharides through hydrolysis. The gene (1650-bp) encoding a novel, bi-modular glycoside hydrolase family 64 (GH64) endo-β-1,3-glucanase (GluY) with a ricin-type β-trefoil lectin domain (RICIN)-like domain from Cellulosimicrobium funkei HY-13 was identified and biocatalytically characterized. The recombinant enzyme (rGluY: 57.5 kDa) displayed the highest degradation activity for laminarin at pH 4.5 and 40 °C, while the polysaccharide was maximally decomposed by its C-terminal truncated mutant enzyme (rGluYΔRICIN: 42.0 kDa) at pH 5.5 and 45 °C. The specific activity (26.0 U/mg) of rGluY for laminarin was 2.6-fold higher than that (9.8 U/mg) of rGluYΔRICIN for the same polysaccharide. Moreover, deleting the C-terminal RICIN domain in the intact enzyme caused a significant decrease (>60%) of its ability to degrade β-1,3-d-glucans such as pachyman and curdlan. Biocatalytic degradation of β-1,3-d-glucans by inverting rGluY yielded predominantly d-laminaripentaose. rGluY exhibited stronger growth inhibition against Candida albicans in a dose-dependent manner than rGluYΔRICIN. The degree of growth inhibition of C. albicans by rGluY (approximately 1.8 μM) was approximately 80% of the fungal growth. The superior anti-fungal activity of rGluY suggests that it can potentially be exploited as a supplementary agent in the food and pharmaceutical industries. Full article

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15 pages, 339 KiB

Open AccessEditor’s ChoiceReview

Neutrophil Elastase and Chronic Lung Disease

by Judith A. Voynow and Meagan Shinbashi

Biomolecules 2021, 11(8), 1065; https://doi.org/10.3390/biom11081065 - 21 Jul 2021

Cited by 67 | Viewed by 10826

Abstract

Neutrophil elastase (NE) is a major inflammatory protease released by neutrophils and is present in the airways of patients with cystic fibrosis (CF), chronic obstructive pulmonary disease, non-CF bronchiectasis, and bronchopulmonary dysplasia. Although NE facilitates leukocyte transmigration to the site of infection and [...] Read more.

Neutrophil elastase (NE) is a major inflammatory protease released by neutrophils and is present in the airways of patients with cystic fibrosis (CF), chronic obstructive pulmonary disease, non-CF bronchiectasis, and bronchopulmonary dysplasia. Although NE facilitates leukocyte transmigration to the site of infection and is required for clearance of Gram-negative bacteria, it also activates inflammation when released into the airway milieu in chronic inflammatory airway diseases. NE exposure induces airway remodeling with increased mucin expression and secretion and impaired ciliary motility. NE interrupts epithelial repair by promoting cellular apoptosis and senescence and it activates inflammation directly by increasing cytokine expression and release, and indirectly by triggering extracellular trap release and exosome release, which magnify protease activity and inflammation in the airway. NE inhibits innate immune function by digesting opsonins and opsonin receptors, degrading innate immune proteins such as lactoferrin, and inhibiting macrophage phagocytosis. Importantly, NE-directed therapies have not yet been effective in preventing the pathologic sequelae of NE exposure, but new therapies are being developed that offer both direct antiprotease activity and multifunctional anti-inflammatory properties. Full article

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15 pages, 1958 KiB

Open AccessEditor’s ChoiceArticle

Credentialing and Pharmacologically Targeting PTP4A3 Phosphatase as a Molecular Target for Ovarian Cancer

by John S. Lazo, Elizabeth R. Sharlow, Robert Cornelison, Duncan J. Hart, Danielle C. Llaneza, Anna J. Mendelson, Ettore J. Rastelli, Nikhil R. Tasker, Charles N. Landen, Jr. and Peter Wipf

Biomolecules 2021, 11(7), 969; https://doi.org/10.3390/biom11070969 - 30 Jun 2021

Cited by 5 | Viewed by 3580

Abstract

High grade serous ovarian cancer (OvCa) frequently becomes drug resistant and often recurs. Consequently, new drug targets and therapies are needed. Bioinformatics-based studies uncovered a relationship between high Protein Tyrosine Phosphatase of Regenerating Liver-3 (PRL3 also known as PTP4A3) expression and poor patient [...] Read more.

High grade serous ovarian cancer (OvCa) frequently becomes drug resistant and often recurs. Consequently, new drug targets and therapies are needed. Bioinformatics-based studies uncovered a relationship between high Protein Tyrosine Phosphatase of Regenerating Liver-3 (PRL3 also known as PTP4A3) expression and poor patient survival in both early and late stage OvCa. PTP4A3 mRNA levels were 5–20 fold higher in drug resistant or high grade serous OvCa cell lines compared to nonmalignant cells. JMS-053 is a potent allosteric small molecule PTP4A3 inhibitor and to explore further the role of PTP4A3 in OvCa, we synthesized and interrogated a series of JMS-053-based analogs in OvCa cell line-based phenotypic assays. While the JMS-053 analogs inhibit in vitro PTP4A3 enzyme activity, none were superior to JMS-053 in reducing high grade serous OvCa cell survival. Because PTP4A3 controls cell migration, we interrogated the effect of JMS-053 on this cancer-relevant process. Both JMS-053 and CRISPR/Cas9 PTP4A3 depletion blocked cell migration. The inhibition caused by JMS-053 required the presence of PTP4A3. JMS-053 caused additive or synergistic in vitro cytotoxicity when combined with paclitaxel and reduced in vivo OvCa dissemination. These results indicate the importance of PTP4A3 in OvCa and support further investigations of the lead inhibitor, JMS-053. Full article

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17 pages, 18183 KiB

Open AccessArticle

A Pan-Inhibitor for Protein Arginine Methyltransferase Family Enzymes

by Iredia D. Iyamu, Ayad A. Al-Hamashi and Rong Huang

Biomolecules 2021, 11(6), 854; https://doi.org/10.3390/biom11060854 - 08 Jun 2021

Cited by 13 | Viewed by 4463

Abstract

Protein arginine methyltransferases (PRMTs) play important roles in transcription, splicing, DNA damage repair, RNA biology, and cellular metabolism. Thus, PRMTs have been attractive targets for various diseases. In this study, we reported the design and synthesis of a potent pan-inhibitor for PRMTs that [...] Read more.

Protein arginine methyltransferases (PRMTs) play important roles in transcription, splicing, DNA damage repair, RNA biology, and cellular metabolism. Thus, PRMTs have been attractive targets for various diseases. In this study, we reported the design and synthesis of a potent pan-inhibitor for PRMTs that tethers a thioadenosine and various substituted guanidino groups through a propyl linker. Compound II757 exhibits a half-maximal inhibition concentration (IC₅₀) value of 5 to 555 nM for eight tested PRMTs, with the highest inhibition for PRMT4 (IC₅₀ = 5 nM). The kinetic study demonstrated that II757 competitively binds at the SAM binding site of PRMT1. Notably, II757 is selective for PRMTs over a panel of other methyltransferases, which can serve as a general probe for PRMTs and a lead for further optimization to increase the selectivity for individual PRMT. Full article

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16 pages, 4010 KiB

Open AccessArticle

Identification and Characterization of a Novel, Cold-Adapted d-Xylobiose- and d-Xylose-Releasing Endo-β-1,4-Xylanase from an Antarctic Soil Bacterium, Duganella sp. PAMC 27433

by Do Young Kim, Jonghoon Kim, Yung Mi Lee, Jong Suk Lee, Dong-Ha Shin, Bon-Hwan Ku, Kwang-Hee Son and Ho-Yong Park

Biomolecules 2021, 11(5), 680; https://doi.org/10.3390/biom11050680 - 30 Apr 2021

Cited by 8 | Viewed by 2252

Abstract

Endo-β-1,4-xylanase is a key enzyme in the degradation of β-1,4-d-xylan polysaccharides through hydrolysis. A glycoside hydrolase family 10 (GH10) endo-β-1,4-xylanase (XylR) from Duganella sp. PAMC 27433, an Antarctic soil bacterium, was identified and functionally characterized. The XylR gene (1122-bp) encoded an [...] Read more.

Endo-β-1,4-xylanase is a key enzyme in the degradation of β-1,4-d-xylan polysaccharides through hydrolysis. A glycoside hydrolase family 10 (GH10) endo-β-1,4-xylanase (XylR) from Duganella sp. PAMC 27433, an Antarctic soil bacterium, was identified and functionally characterized. The XylR gene (1122-bp) encoded an acidic protein containing a single catalytic GH10 domain that was 86% identical to that of an uncultured bacterium BLR13 endo-β-1,4-xylanase (ACN58881). The recombinant enzyme (rXylR: 42.0 kDa) showed the highest beechwood xylan-degrading activity at pH 5.5 and 40 °C, and displayed 12% of its maximum activity even at 4 °C. rXylR was not only almost completely inhibited by 5 mM N-bromosuccinimide or metal ions (each 1 mM) including Hg²⁺, Ca²⁺, or Cu²⁺ but also significantly suppressed by 1 mM Ni²⁺, Zn²⁺, or Fe²⁺. However, its enzyme activity was upregulated (>1.4-fold) in the presence of 0.5% Triton X-100 or Tween 80. The specific activities of rXylR toward beechwood xylan, birchwood xylan, oat spelts xylan, and p-nitrophenyl-β-d-cellobioside were 274.7, 103.2, 35.6, and 365.1 U/mg, respectively. Enzymatic hydrolysis of birchwood xylan and d-xylooligosaccharides yielded d-xylose and d-xylobiose as the end products. The results of the present study suggest that rXylR is a novel cold-adapted d-xylobiose- and d-xylose-releasing endo-β-1,4-xylanase. Full article

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23 pages, 3835 KiB

Open AccessArticle

Novel Chemically Modified Curcumin (CMC) Derivatives Inhibit Tyrosinase Activity and Melanin Synthesis in B16F10 Mouse Melanoma Cells

by Shilpi Goenka, Francis Johnson and Sanford R. Simon

Biomolecules 2021, 11(5), 674; https://doi.org/10.3390/biom11050674 - 30 Apr 2021

Cited by 10 | Viewed by 3376

Abstract

Skin hyperpigmentation disorders arise due to excessive production of the macromolecular pigment melanin catalyzed by the enzyme tyrosinase. Recently, the therapeutic use of curcumin for inhibiting tyrosinase activity and production of melanin have been recognized, but poor stability and solubility have limited its [...] Read more.

Skin hyperpigmentation disorders arise due to excessive production of the macromolecular pigment melanin catalyzed by the enzyme tyrosinase. Recently, the therapeutic use of curcumin for inhibiting tyrosinase activity and production of melanin have been recognized, but poor stability and solubility have limited its use, which has inspired synthesis of curcumin analogs. Here, we investigated four novel chemically modified curcumin (CMC) derivatives (CMC2.14, CMC2.5, CMC2.23 and CMC2.24) and compared them to the parent compound curcumin (PC) for inhibition of in vitro tyrosinase activity using two substrates for monophenolase and diphenolase activities of the enzyme and for diminution of cellular melanogenesis. Enzyme kinetics were analyzed using Lineweaver-Burk and Dixon plots and nonlinear curve-fitting to determine the mechanism for tyrosinase inhibition. Copper chelating activity, using pyrocatechol violet dye indicator assay, and antioxidant activity, using a DPPH radical scavenging assay, were also conducted. Next, the capacity of these derivatives to inhibit tyrosinase-catalyzed melanogenesis was studied in B16F10 mouse melanoma cells and the mechanisms of inhibition were elucidated. Inhibition mechanisms were studied by measuring intracellular tyrosinase activity, cell-free and intracellular α-glucosidase enzyme activity, and effects on MITF protein level and cAMP maturation factor. Our results showed that CMC2.24 showed the greatest efficacy as a tyrosinase inhibitor of all the CMCs and was better than PC as well as a popular tyrosinase inhibitor-kojic acid. Both CMC2.24 and CMC2.23 inhibited tyrosinase enzyme activity by a mixed mode of inhibition with a predominant competitive mode. In addition, CMC2.24 as well as CMC2.23 showed a comparable robust efficacy in inhibiting melanogenesis in cultured melanocytes. Furthermore, after removal of CMC2.24 or CMC2.23 from the medium, we could demonstrate a partial recovery of the suppressed intracellular tyrosinase activity in the melanocytes. Our results provide a proof-of-principle for the novel use of the CMCs that shows them to be far superior to the parent compound, curcumin, for skin depigmentation. Full article

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16 pages, 6465 KiB

Open AccessArticle

Conformational Plasticity-Rigidity Axis of the Coagulation Factor VII Zymogen Elucidated by Atomistic Simulations of the N-Terminally Truncated Factor VIIa Protease Domain

by Jesper J. Madsen and Ole H. Olsen

Biomolecules 2021, 11(4), 549; https://doi.org/10.3390/biom11040549 - 08 Apr 2021

Cited by 4 | Viewed by 2672

Abstract

The vast majority of coagulation factor VII (FVII), a trypsin-like protease, circulates as the inactive zymogen. Activated FVII (FVIIa) is formed upon proteolytic activation of FVII, where it remains in a zymogen-like state and it is fully activated only when bound to tissue [...] Read more.

The vast majority of coagulation factor VII (FVII), a trypsin-like protease, circulates as the inactive zymogen. Activated FVII (FVIIa) is formed upon proteolytic activation of FVII, where it remains in a zymogen-like state and it is fully activated only when bound to tissue factor (TF). The catalytic domains of trypsin-like proteases adopt strikingly similar structures in their fully active forms. However, the dynamics and structures of the available corresponding zymogens reveal remarkable conformational plasticity of the protease domain prior to activation in many cases. Exactly how ligands and cofactors modulate the conformational dynamics and function of these proteases is not entirely understood. Here, we employ atomistic simulations of FVIIa (and variants hereof, including a TF-independent variant and N-terminally truncated variants) to provide fundamental insights with atomistic resolution into the plasticity-rigidity interplay of the protease domain conformations that appears to govern the functional response to proteolytic and allosteric activation. We argue that these findings are relevant to the FVII zymogen, whose structure has remained elusive despite substantial efforts. Our results shed light on the nature of FVII and demonstrate how conformational dynamics has played a crucial role in the evolutionary adaptation of regulatory mechanisms that were not present in the ancestral trypsin. Exploiting this knowledge could lead to engineering of protease variants for use as next-generation hemostatic therapeutics. Full article

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14 pages, 15453 KiB

Open AccessArticle

Substrate-Dependent Sensitivity of SIRT1 to Nicotinamide Inhibition

by Stacia Rymarchyk, Wenjia Kang and Yana Cen

Biomolecules 2021, 11(2), 312; https://doi.org/10.3390/biom11020312 - 18 Feb 2021

Cited by 6 | Viewed by 3284

Abstract

SIRT1 is the most extensively studied human sirtuin with a broad spectrum of endogenous targets. It has been implicated in the regulation of a myriad of cellular events, such as gene transcription, mitochondria biogenesis, insulin secretion as well as glucose and lipid metabolism. [...] Read more.

SIRT1 is the most extensively studied human sirtuin with a broad spectrum of endogenous targets. It has been implicated in the regulation of a myriad of cellular events, such as gene transcription, mitochondria biogenesis, insulin secretion as well as glucose and lipid metabolism. From a mechanistic perspective, nicotinamide (NAM), a byproduct of a sirtuin-catalyzed reaction, reverses a reaction intermediate to regenerate NAD⁺ through “base exchange”, leading to the inhibition of the forward deacetylation. NAM has been suggested as a universal sirtuin negative regulator. Sirtuins have evolved different strategies in response to NAM regulation. Here, we report the detailed kinetic analysis of SIRT1-catalyzed reactions using endogenous substrate-based synthetic peptides. A novel substrate-dependent sensitivity of SIRT1 to NAM inhibition was observed. Additionally, SIRT1 demonstrated pH-dependent deacetylation with normal solvent isotope effects (SIEs), consistent with proton transfer in the rate-limiting step. Base exchange, in contrast, was insensitive to pH changes with no apparent SIEs, indicative of lack of proton transfer in the rate-limiting step. Consequently, NAM inhibition was attenuated at a high pH in proteated buffers. Our study provides new evidence for “activation by de-repression” as an effective sirtuin activation strategy. Full article

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