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21 pages, 5476 KiB

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Synthesis, In Silico and Kinetics Evaluation of N-(β-d-glucopyranosyl)-2-arylimidazole-4(5)-carboxamides and N-(β-d-glucopyranosyl)-4(5)-arylimidazole-2-carboxamides as Glycogen Phosphorylase Inhibitors

by Levente Homolya, Rachel T. Mathomes, Luca Varga, Tibor Docsa, László Juhász, Joseph M. Hayes and László Somsák

Int. J. Mol. Sci. 2024, 25(9), 4591; https://doi.org/10.3390/ijms25094591 - 23 Apr 2024

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Abstract

Recently studied N-(β-d-glucopyranosyl)-3-aryl-1,2,4-triazole-5-carboxamides have proven to be low micromolar inhibitors of glycogen phosphorylase (GP), a validated target for the treatment of type 2 diabetes mellitus. Since in other settings, the bioisosteric replacement of the 1,2,4-triazole moiety with imidazole resulted [...] Read more.

Recently studied N-(β-d-glucopyranosyl)-3-aryl-1,2,4-triazole-5-carboxamides have proven to be low micromolar inhibitors of glycogen phosphorylase (GP), a validated target for the treatment of type 2 diabetes mellitus. Since in other settings, the bioisosteric replacement of the 1,2,4-triazole moiety with imidazole resulted in significantly more efficient GP inhibitors, in silico calculations using Glide molecular docking along with unbound state DFT calculations were performed on N-(β-d-glucopyranosyl)-arylimidazole-carboxamides, revealing their potential for strong GP inhibition. The syntheses of the target compounds involved the formation of an amide bond between per-O-acetylated β-d-glucopyranosylamine and the corresponding arylimidazole-carboxylic acids. Kinetics experiments on rabbit muscle GPb revealed low micromolar inhibitors, with the best inhibition constants (K_is) of ~3–4 µM obtained for 1- and 2-naphthyl-substituted N-(β-d-glucopyranosyl)-imidazolecarboxamides, 2b–c. The predicted protein–ligand interactions responsible for the observed potencies are discussed and will facilitate the structure-based design of other inhibitors targeting this important therapeutic target. Meanwhile, the importance of the careful consideration of ligand tautomeric states in binding calculations is highlighted, with the usefulness of DFT calculations in this regard proposed. Full article

(This article belongs to the Special Issue 21st Century Enzymology: Further Advances in Identification and Characterization of Enzymes)

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16 pages, 5092 KiB

Open AccessArticle

Functional Characterization of Endo- and Exo-Hydrolase Genes in Arabinan Degradation Gene Cluster of Bifidobacterium longum subsp. suis

by Yewon Kang, Chang-Yun Choi, Jihun Kang, Ye-Rin Ju, Hye Bin Kim, Nam Soo Han and Tae-Jip Kim

Int. J. Mol. Sci. 2024, 25(6), 3175; https://doi.org/10.3390/ijms25063175 - 09 Mar 2024

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Abstract

Bifidobacteria are probiotic microorganisms commonly found in the gastrointestinal tract, some of which are known to utilize linear arabino-oligosaccharides (AOS) as prebiotic carbohydrates. In general, the synergistic actions of exo-type α-l-arabinofuranosidases (ABFs) and endo-α-1,5-l-arabinanases (ABNs) are required for efficient [...] Read more.

Bifidobacteria are probiotic microorganisms commonly found in the gastrointestinal tract, some of which are known to utilize linear arabino-oligosaccharides (AOS) as prebiotic carbohydrates. In general, the synergistic actions of exo-type α-l-arabinofuranosidases (ABFs) and endo-α-1,5-l-arabinanases (ABNs) are required for efficient arabinan degradation. In this study, the putative gene cluster for arabinan degradation was discovered in the genome of Bifidobacterium longum subsp. suis. It consists of a variety of genes encoding exo- and endo-hydrolases, sugar-binding proteins, ABC-binding cassettes, and transcriptional regulators. Among them, two endo-ABNs GH43 (BflsABN43A and BflsABN43B), two exo-ABFs GH43 (BflsABF43A and BflsABF43B), and an exo-ABF GH51 (BflsABF51) were predicted to be the key hydrolases for arabinan degradation. These hydrolase genes were functionally expressed in Escherichia coli, and their enzymatic properties were characterized. Their synergism in arabinan degradation has been proposed from the detailed modes of action. Extracellular endo-BflsABN43A hydrolyzes sugar beet and debranched arabinans into the short-chain branched and linear AOS. Intracellularly, AOS can be further degraded into l-arabinose via the cooperative actions of endo-BflsABN43B, exo-BflsABF43A with debranching activity, α-1,5-linkage-specific exo-BflsABF43B, and exo-BflsABF51 with dual activities. The resulting l-arabinose is expected to be metabolized into energy through the pentose phosphate pathway by three enzymes expressed from the ara operon of bifidobacteria. It is anticipated that uncovering arabinan utilization gene clusters and their detailed functions in the genomes of diverse microorganisms will facilitate the development of customized synbiotics. Full article

(This article belongs to the Special Issue 21st Century Enzymology: Further Advances in Identification and Characterization of Enzymes)

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13 pages, 2120 KiB

Open AccessArticle

Biodegradation of Polystyrene by Galleria mellonella: Identification of Potential Enzymes Involved in the Degradative Pathway

by Sebastián Venegas, Carolina Alarcón, Juan Araya, Marcell Gatica, Violeta Morin, Estefanía Tarifeño-Saldivia and Elena Uribe

Int. J. Mol. Sci. 2024, 25(3), 1576; https://doi.org/10.3390/ijms25031576 - 27 Jan 2024

Cited by 1 | Viewed by 1138

Abstract

Galleria mellonella is a lepidopteran whose larval stage has shown the ability to degrade polystyrene (PS), one of the most recalcitrant plastics to biodegradation. In the present study, we fed G. mellonella larvae with PS for 54 days and determined candidate enzymes for [...] Read more.

Galleria mellonella is a lepidopteran whose larval stage has shown the ability to degrade polystyrene (PS), one of the most recalcitrant plastics to biodegradation. In the present study, we fed G. mellonella larvae with PS for 54 days and determined candidate enzymes for its degradation. We first confirmed the biodegradation of PS by Fourier transform infrared spectroscopy- Attenuated total reflectance (FTIR-ATR) and then identified candidate enzymes in the larval gut by proteomic analysis using liquid chromatography with tandem mass spectrometry (LC-MS/MS). Two of these proteins have structural similarities to the styrene-degrading enzymes described so far. In addition, potential hydrolases, isomerases, dehydrogenases, and oxidases were identified that show little similarity to the bacterial enzymes that degrade styrene. However, their response to a diet based solely on polystyrene makes them interesting candidates as a potential new group of polystyrene-metabolizing enzymes in eukaryotes. Full article

(This article belongs to the Special Issue 21st Century Enzymology: Further Advances in Identification and Characterization of Enzymes)

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13 pages, 4755 KiB

Open AccessArticle

Charged Amino Acids in the Transmembrane Helix Strongly Affect the Enzyme Activity of Aromatase

by Juliane Günther, Gerhard Schuler, Elin Teppa and Rainer Fürbass

Int. J. Mol. Sci. 2024, 25(3), 1440; https://doi.org/10.3390/ijms25031440 - 24 Jan 2024

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Abstract

Estrogens play critical roles in embryonic development, gonadal sex differentiation, behavior, and reproduction in vertebrates and in several human cancers. Estrogens are synthesized from testosterone and androstenedione by the endoplasmic reticulum membrane-bound P450 aromatase/cytochrome P450 oxidoreductase complex (CYP19/CPR). Here, we report the characterization [...] Read more.

Estrogens play critical roles in embryonic development, gonadal sex differentiation, behavior, and reproduction in vertebrates and in several human cancers. Estrogens are synthesized from testosterone and androstenedione by the endoplasmic reticulum membrane-bound P450 aromatase/cytochrome P450 oxidoreductase complex (CYP19/CPR). Here, we report the characterization of novel mammalian CYP19 isoforms encoded by CYP19 gene copies. These CYP19 isoforms are all defined by a combination of mutations in the N-terminal transmembrane helix (E42K, D43N) and in helix C of the catalytic domain (P146T, F147Y). The mutant CYP19 isoforms show increased androgen conversion due to the KN transmembrane helix. In addition, the TY substitutions in helix C result in a substrate preference for androstenedione. Our structural models suggest that CYP19 mutants may interact differently with the membrane (affecting substrate uptake) and with CPR (affecting electron transfer), providing structural clues for the catalytic differences. Full article

(This article belongs to the Special Issue 21st Century Enzymology: Further Advances in Identification and Characterization of Enzymes)

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16 pages, 7839 KiB

Open AccessArticle

Can Activation of Acetylcholinesterase by β-Amyloid Peptide Decrease the Effectiveness of Cholinesterase Inhibitors?

by Irina V. Zueva, Elmira A. Vasilieva, Gulnara A. Gaynanova, Andrey V. Moiseenko, Anna D. Burtseva, Konstantin M. Boyko, Lucia Ya. Zakharova and Konstantin A. Petrov

Int. J. Mol. Sci. 2023, 24(22), 16395; https://doi.org/10.3390/ijms242216395 - 16 Nov 2023

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Abstract

A central event in the pathogenesis of Alzheimer’s disease (AD) is the accumulation of senile plaques composed of aggregated amyloid-β (Aβ) peptides. The main class of drugs currently used for the treatment of AD are the acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) inhibitors. In [...] Read more.

A central event in the pathogenesis of Alzheimer’s disease (AD) is the accumulation of senile plaques composed of aggregated amyloid-β (Aβ) peptides. The main class of drugs currently used for the treatment of AD are the acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) inhibitors. In this study, it has been shown that Aβ augmented AChE activity in vitro, maximum activation of 548 ± 5% was achieved following 48 h of incubation with 10 μM of Aβ_1–40, leading to a 7.7-fold increase in catalytic efficiency. The observed non-competitive type of AChE activation by Aβ_1–40 was associated with increased V_max and unchanged K_m. Although BChE activity also increased following incubation with Aβ_1–40, this was less efficiently achieved as compared with AChE. Ex vivo electrophysiological experiments showed that 10 μM of Aβ_1–40 significantly decreased the effect of the AChE inhibitor huperzine A on the synaptic potential parameters. Full article

(This article belongs to the Special Issue 21st Century Enzymology: Further Advances in Identification and Characterization of Enzymes)

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13 pages, 2825 KiB

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Changing the Electron Acceptor Specificity of Rhodobacter capsulatus Formate Dehydrogenase from NAD⁺ to NADP⁺

by Hemant Kumar and Silke Leimkühler

Int. J. Mol. Sci. 2023, 24(22), 16067; https://doi.org/10.3390/ijms242216067 - 08 Nov 2023

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Abstract

Formate dehydrogenases catalyze the reversible oxidation of formate to carbon dioxide. These enzymes play an important role in CO₂ reduction and serve as nicotinamide cofactor recycling enzymes. More recently, the CO₂-reducing activity of formate dehydrogenases, especially metal-containing formate dehydrogenases, has [...] Read more.

Formate dehydrogenases catalyze the reversible oxidation of formate to carbon dioxide. These enzymes play an important role in CO₂ reduction and serve as nicotinamide cofactor recycling enzymes. More recently, the CO₂-reducing activity of formate dehydrogenases, especially metal-containing formate dehydrogenases, has been further explored for efficient atmospheric CO₂ capture. Here, we investigate the nicotinamide binding site of formate dehydrogenase from Rhodobacter capsulatus for its specificity toward NAD⁺ vs. NADP⁺ reduction. Starting from the NAD⁺-specific wild-type RcFDH, key residues were exchanged to enable NADP⁺ binding on the basis of the NAD⁺-bound cryo-EM structure (PDB-ID: 6TG9). It has been observed that the lysine at position 157 (Lys¹⁵⁷) in the β-subunit of the enzyme is essential for the binding of NAD⁺. RcFDH variants that had Glu²⁵⁹ exchanged for either a positively charged or uncharged amino acid had additional activity with NADP⁺. The FdsB^L279R and FdsB^K276A variants also showed activity with NADP⁺. Kinetic parameters for all the variants were determined and tested for activity in CO₂ reduction. The variants were able to reduce CO₂ using NADPH as an electron donor in a coupled assay with phosphite dehydrogenase (PTDH), which regenerates NADPH. This makes the enzyme suitable for applications where it can be coupled with other enzymes that use NADPH. Full article

(This article belongs to the Special Issue 21st Century Enzymology: Further Advances in Identification and Characterization of Enzymes)

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13 pages, 2750 KiB

Open AccessArticle

Expression and Characterization of a β-Galactosidase from the Pacific Oyster, Crassostrea gigas, and Evaluation of Strategies for Testing Substrate Specificity

by Julia Thoma, Reingard Grabherr and Erika Staudacher

Int. J. Mol. Sci. 2023, 24(20), 15287; https://doi.org/10.3390/ijms242015287 - 18 Oct 2023

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Abstract

β-Galactosidases (EC 3.2.1.23) are exoglycosidases that catalyze the cleavage of glycoconjugates with terminal β-D-galactose residues in β1,3-, β1,4- or β1,6-linkage. Although this family of exoglycosidases has been extensively studied in vertebrates, plants, yeast, and bacteria, little information is available for mollusks. Mollusks are [...] Read more.

β-Galactosidases (EC 3.2.1.23) are exoglycosidases that catalyze the cleavage of glycoconjugates with terminal β-D-galactose residues in β1,3-, β1,4- or β1,6-linkage. Although this family of exoglycosidases has been extensively studied in vertebrates, plants, yeast, and bacteria, little information is available for mollusks. Mollusks are a diverse and highly successful group of animals that play many different roles in their ecosystems, including filter feeders and detritivores. Here, the first β-galactosidase from the Pacific oyster, Crassostrea gigas was discovered, biochemically characterized, and compared to our previously characterized slug enzyme from Arion vulgaris (UniProt Ref. Nr.: A0A0B7AQJ9). Overall, the mussel enzyme showed similar biochemical parameters to the snail enzyme. The enzyme from C. gigas was most active in an acidic environment (pH 3.5) and at a reaction temperature of 50 °C. Optimal storage conditions were up to 37 °C. In contrast to the enzyme from A. vulgaris, the supplementation of cations (Ni²⁺, Co²⁺, Mn²⁺, Mg²⁺, Ca²⁺, Cu²⁺, Ba²⁺) increased the activity of the enzyme from C. gigas. Substrate specificity studies of the β-galactosidases from the mussel, C. gigas, and the slug, A. vulgaris, revealed activity towards terminal β1,3- and β1,4-linked galactose residues for both enzymes. Using the same substrates in labeled and unlabeled form, we were able to detect the effect of labeling on the β-galactosidase activity using MALDI-TOF MS, HPTLC, and HPLC. While lactose was cleaved by the enzymes in an unlabeled or labeled state, galacto-N-biose was not cleaved as soon as a 2-amino benzoic acid label was added. In this study we present the biochemical characterization of the first recombinantly expressed β-galactosidase from the Pacific oyster, C. gigas, and we compare different analytical methods for the determination of β-galactosidase activity using the enzyme from C. gigas and A. vulgaris. Full article

(This article belongs to the Special Issue 21st Century Enzymology: Further Advances in Identification and Characterization of Enzymes)

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15 pages, 3219 KiB

Open AccessArticle

Biochemical and Structural Insights into a Thiamine Diphosphate-Dependent α-Ketoglutarate Decarboxylase from Cyanobacterium Microcystis aeruginosa NIES-843

by Zhi-Min Li, Ziwei Hu, Xiaoqin Wang, Suhang Chen, Weiyan Yu, Jianping Liu and Zhimin Li

Int. J. Mol. Sci. 2023, 24(15), 12198; https://doi.org/10.3390/ijms241512198 - 30 Jul 2023

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Abstract

α-Ketoglutarate decarboxylase is a crucial enzyme in the tricarboxylic acid cycle of cyanobacteria, catalyzing the non-oxidative decarboxylation of α-ketoglutarate to produce succinate semialdehyde and CO₂. The decarboxylation process is reliant on the cofactor of thiamine diphosphate. However, this enzyme’s biochemical and [...] Read more.

α-Ketoglutarate decarboxylase is a crucial enzyme in the tricarboxylic acid cycle of cyanobacteria, catalyzing the non-oxidative decarboxylation of α-ketoglutarate to produce succinate semialdehyde and CO₂. The decarboxylation process is reliant on the cofactor of thiamine diphosphate. However, this enzyme’s biochemical and structural properties have not been well characterized. In this work, two α-ketoglutarate decarboxylases encoded by MAE_06010 and MiAbw_01735 genes from Microcystis aeruginosa NIES-843 (MaKGD) and NIES-4325 (MiKGD), respectively, were overexpressed and purified by using an Escherichia coli expression system. It was found that MaKGD exhibited 9.2-fold higher catalytic efficiency than MiKGD, which may be attributed to the absence of glutamate decarboxylase in Microcystis aeruginosa NIES-843. Further biochemical investigation of MaKGD demonstrated that it displayed optimum activity at pH 6.5–7.0 and was most activated by Mg²⁺. Additionally, MaKGD showed substrate specificity towards α-ketoglutarate. Structural modeling and autodocking results revealed that the active site of MaKGD contained a distinct binding pocket where α-ketoglutarate and thiamine diphosphate interacted with specific amino acid residues via hydrophobic interactions, hydrogen bonds and salt bridges. Furthermore, the mutagenesis study provided strong evidence supporting the importance of certain residues in the catalysis of MaKGD. These findings provide new insights into the structure-function relationships of α-ketoglutarate decarboxylases from cyanobacteria. Full article

(This article belongs to the Special Issue 21st Century Enzymology: Further Advances in Identification and Characterization of Enzymes)

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15 pages, 2181 KiB

Open AccessArticle

De Novo Computational Design of a Lipase with Hydrolysis Activity towards Middle-Chained Fatty Acid Esters

by Jinsha Huang, Xiaoman Xie, Zhen Zheng, Luona Ye, Pengbo Wang, Li Xu, Ying Wu, Jinyong Yan, Min Yang and Yunjun Yan

Int. J. Mol. Sci. 2023, 24(10), 8581; https://doi.org/10.3390/ijms24108581 - 11 May 2023

Cited by 2 | Viewed by 1881

Abstract

Innovations in biocatalysts provide great prospects for intolerant environments or novel reactions. Due to the limited catalytic capacity and the long-term and labor-intensive characteristics of mining enzymes with the desired functions, de novo enzyme design was developed to obtain industrial application candidates in [...] Read more.

Innovations in biocatalysts provide great prospects for intolerant environments or novel reactions. Due to the limited catalytic capacity and the long-term and labor-intensive characteristics of mining enzymes with the desired functions, de novo enzyme design was developed to obtain industrial application candidates in a rapid and convenient way. Here, based on the catalytic mechanisms and the known structures of proteins, we proposed a computational protein design strategy combining de novo enzyme design and laboratory-directed evolution. Starting with the theozyme constructed using a quantum-mechanical approach, the theoretical enzyme-skeleton combinations were assembled and optimized via the Rosetta “inside-out” protocol. A small number of designed sequences were experimentally screened using SDS-PAGE, mass spectrometry and a qualitative activity assay in which the designed enzyme 1a8uD₁ exhibited a measurable hydrolysis activity of 24.25 ± 0.57 U/g towards p-nitrophenyl octanoate. To improve the activity of the designed enzyme, molecular dynamics simulations and the RosettaDesign application were utilized to further optimize the substrate binding mode and amino acid sequence, thus keeping the residues of theozyme intact. The redesigned lipase 1a8uD₁–M8 displayed enhanced hydrolysis activity towards p-nitrophenyl octanoate—3.34 times higher than that of 1a8uD₁. Meanwhile, the natural skeleton protein (PDB entry 1a8u) did not display any hydrolysis activity, confirming that the hydrolysis abilities of the designed 1a8uD₁ and the redesigned 1a8uD₁–M8 were devised from scratch. More importantly, the designed 1a8uD₁–M8 was also able to hydrolyze the natural middle-chained substrate (glycerol trioctanoate), for which the activity was 27.67 ± 0.69 U/g. This study indicates that the strategy employed here has great potential to generate novel enzymes exhibiting the desired reactions. Full article

(This article belongs to the Special Issue 21st Century Enzymology: Further Advances in Identification and Characterization of Enzymes)

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18 pages, 2728 KiB

Open AccessArticle

Kinetic and Regulatory Properties of Yarrowia lipolytica Aconitate Hydratase as a Model-Indicator of Cell Redox State under pH Stress

by Tatyana I. Rakhmanova, Varvara Yu. Sekova, Natalya N. Gessler, Elena P. Isakova, Yulia I. Deryabina, Tatyana N. Popova, Yevgeniya I. Shurubor and Boris F. Krasnikov

Int. J. Mol. Sci. 2023, 24(8), 7670; https://doi.org/10.3390/ijms24087670 - 21 Apr 2023

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Abstract

This paper presents an analysis of the regulation activity of the partially purified preparations of cellular aconitate hydratase (AH) on the yeast Yarrowia lipolytica cultivated at extreme pH. As a result of purification, enzyme preparations were obtained from cells grown on media at [...] Read more.

This paper presents an analysis of the regulation activity of the partially purified preparations of cellular aconitate hydratase (AH) on the yeast Yarrowia lipolytica cultivated at extreme pH. As a result of purification, enzyme preparations were obtained from cells grown on media at pH 4.0, 5.5, and 9.0, purified by 48-, 46-, and 51-fold and having a specific activity of 0.43, 0.55 and 0.36 E/mg protein, respectively. The kinetic parameters of preparations from cells cultured at extreme pH demonstrated: (1) an increase in the affinity for citrate and isocitrate; and (2) a shift in the pH optima to the acidic and alkaline side in accordance with the modulation of the medium pH. The regulatory properties of the enzyme from cells subjected to alkaline stress showed increased sensitivity to Fe²⁺ ions and high peroxide resistance. Reduced glutathione (GSH) stimulated AH, while oxidized glutathione (GSSG) inhibited AH. A more pronounced effect of both GSH and GSSG was noted for the enzyme obtained from cells grown at pH 5.5. The data obtained provide new approaches to the use of Y. lipolytica as a model of eukaryotic cells demonstrating the development of a stress-induced pathology and to conducting a detailed analysis of enzymatic activity for its correction. Full article

(This article belongs to the Special Issue 21st Century Enzymology: Further Advances in Identification and Characterization of Enzymes)

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11 pages, 930 KiB

Open AccessArticle

Expanding the Use of Peroxygenase from Oat Flour in Organic Synthesis: Enantioselective Oxidation of Sulfides

by Claudia Sanfilippo, Federica Cernuto and Angela Patti

Int. J. Mol. Sci. 2023, 24(8), 7464; https://doi.org/10.3390/ijms24087464 - 18 Apr 2023

Cited by 1 | Viewed by 970

Abstract

Biocatalyzed oxidations are an important target in sustainable synthesis since chemical oxidations often require harsh conditions and metal-based catalysts. A raw peroxygenase-containing enzymatic preparation from oat flour was tested as a biocatalyst for the enantioselective oxidation of sulfides to sulfoxides and the variations [...] Read more.

Biocatalyzed oxidations are an important target in sustainable synthesis since chemical oxidations often require harsh conditions and metal-based catalysts. A raw peroxygenase-containing enzymatic preparation from oat flour was tested as a biocatalyst for the enantioselective oxidation of sulfides to sulfoxides and the variations of some reaction parameters were evaluated. Under optimal conditions, thioanisole was fully converted into the corresponding (R)-sulfoxide with high optical purity (80% ee) and the same stereopreference was maintained in the oxidation of some other sulfides. Changes in the substituent on the sulfur atom affected the selectivity of the enzyme and the best results were obtained with phenyl methoxymethyl sulfide, which gave the corresponding sulfoxide in 92% ee as exclusive product. The over-oxidation of sulfides to sulfones was instead detected in all the other cases and preferential oxidation of the (S)-enantiomer of the sulfoxide intermediate was observed, albeit with low selectivity. Carrying out the oxidation of thioanisole up to the 29% formation of sulfone led to enhancement of the sulfoxide optical purity (89% ee). The activity in sulfoxidation reactions, in addition to that reported in the epoxidation of different substrates, makes this plant peroxygenase a promising and useful tool in organic synthesis. Full article

(This article belongs to the Special Issue 21st Century Enzymology: Further Advances in Identification and Characterization of Enzymes)

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11 pages, 2553 KiB

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How Single Amino Acid Substitutions Can Disrupt a Protein Hetero-Dimer Interface: Computational and Experimental Studies of the LigAB Dioxygenase from Sphingobium sp. Strain SYK-6

by Angelika Rafalowski, Bakar A. Hassan, Kate Lou, Minh Chau Nguyen and Erika A. Taylor

Int. J. Mol. Sci. 2023, 24(7), 6319; https://doi.org/10.3390/ijms24076319 - 28 Mar 2023

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Abstract

Protocatechuate 4,5-dioxygenase (LigAB) is a heterodimeric enzyme that catalyzes the dioxygenation of multiple lignin derived aromatic compounds. The active site of LigAB is at the heterodimeric interface, with specificity conferred by the alpha subunit and catalytic residues contributed by the beta subunit. Previous [...] Read more.

Protocatechuate 4,5-dioxygenase (LigAB) is a heterodimeric enzyme that catalyzes the dioxygenation of multiple lignin derived aromatic compounds. The active site of LigAB is at the heterodimeric interface, with specificity conferred by the alpha subunit and catalytic residues contributed by the beta subunit. Previous research has indicated that the phenylalanine at the 103 position of the alpha subunit (F103α) controls selectivity for the C5 position of the aromatic substrates, and mutations of this residue can enhance the rate of catalysis for substrates with larger functional groups at this position. While several of the mutations to this position (Valine, V; Threonine, T; Leucine, L; and Histidine, H) were catalytically active, other mutations (Alanine, A; and Serine, S) were found to have reduced dimer interface affinity, leading to challenges in copurifing the catalytically active enzyme complex under high salt conditions. In this study, we aimed to experimentally and computationally interrogate residues at the dimer interface to discern the importance of position 103α for maintaining the integrity of the heterodimer. Molecular dynamic simulations and electrophoretic mobility assays revealed a preference for nonpolar/aromatic amino acids in this position, suggesting that while substitutions to polar amino acids may produce a dioxygenase with a useful substrate utilization profile, those considerations may be off-set by potential destabilization of the catalytically active oligomer. Understanding the dimerization of LigAB provides insight into the multimeric proteins within the largely uncharacterized superfamily and characteristics to consider when engineering proteins that can degrade lignin efficiently. These results shed light on the challenges associated with engineering proteins for broader substrate specificity. Full article

(This article belongs to the Special Issue 21st Century Enzymology: Further Advances in Identification and Characterization of Enzymes)

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15 pages, 4195 KiB

Open AccessArticle

5-Aminolevrinic Acid Exhibits Dual Effects on Stemness in Human Sarcoma Cell Lines under Dark Conditions

by Shohei Horii, Shiori Mori, Ruiko Ogata, Shota Nukaga, Ryoichi Nishida, Shingo Kishi, Rika Sasaki, Ayaka Ikemoto, Takuya Owari, Fumisato Maesaka, Kanya Honoki, Makito Miyake, Yasuhito Tanaka, Kiyohide Fujimoto, Rina Fujiwara-Tani and Hiroki Kuniyasu

Int. J. Mol. Sci. 2023, 24(7), 6189; https://doi.org/10.3390/ijms24076189 - 24 Mar 2023

Cited by 2 | Viewed by 1168

Abstract

5-aminolevulinic acid (ALA) is used for tumor-targeting phototherapy because it is converted to protoporphyrin IX (PPIX) upon excitation and induces phototoxicity. However, the effect of ALA on malignant cells under unexcited conditions is unclear. This information is essential when administering ALA systemically. We [...] Read more.

5-aminolevulinic acid (ALA) is used for tumor-targeting phototherapy because it is converted to protoporphyrin IX (PPIX) upon excitation and induces phototoxicity. However, the effect of ALA on malignant cells under unexcited conditions is unclear. This information is essential when administering ALA systemically. We used sarcoma cell lines that usually arise deep in the body and are rarely exposed to light to examine the effects of ALA treatment under light (daylight lamp irradiation) and dark (dark room) conditions. ALA-treated human SW872 liposarcoma cells and human MG63 osteosarcoma cells cultured under light exhibited growth suppression and increased oxidative stress, while cells cultured in the dark showed no change. However, sphere-forming ability increased in the dark, and the expression of stem-cell-related genes was induced in dark, but not light, conditions. ALA administration increased heme oxygenase 1 (HO-1) expression in both cell types; when carbon monoxide (CO), a metabolite of HO-1, was administered to sarcoma cells via carbon-monoxide-releasing molecule 2 (CORM2), it enhanced sphere-forming ability. We also compared the concentration of biliverdin (BVD) (a co-product of HO-1 activity alongside CO) with sphere-forming ability when HO-1 activity was inhibited using ZnPPIX in the dark. Both cell types showed a peak in sphere-forming ability at 60–80 μM BVD. Furthermore, a cell death inhibitor assay revealed that the HO-1-induced suppression of sphere formation was rescued by apoptosis or ferroptosis inhibitors. These findings suggest that in the absence of excitation, ALA promotes HO-1 expression and enhances the stemness of sarcoma cells, although excessive HO-1 upregulation induces apoptosis and ferroptosis. Our data indicate that systemic ALA administration induces both enhanced stemness and cell death in malignant cells located in dark environments deep in the body and highlight the need to pay attention to drug delivery and ALA concentrations during phototherapy. Full article

(This article belongs to the Special Issue 21st Century Enzymology: Further Advances in Identification and Characterization of Enzymes)

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25 pages, 3785 KiB

Open AccessArticle

The Impact of N^ε-Acryloyllysine Piperazides on the Conformational Dynamics of Transglutaminase 2

by Andreas Heerwig, Alfred Kick, Paul Sommerfeld, Sophia Eimermacher, Frederick Hartung, Markus Laube, Dietmar Fischer, Hans-Jürgen Pietzsch, Jens Pietzsch, Reik Löser, Michael Mertig, Markus Pietsch and Robert Wodtke

Int. J. Mol. Sci. 2023, 24(2), 1650; https://doi.org/10.3390/ijms24021650 - 13 Jan 2023

Cited by 2 | Viewed by 2023

Abstract

In addition to the classic functions of proteins, such as acting as a biocatalyst or binding partner, the conformational states of proteins and their remodeling upon stimulation need to be considered. A prominent example of a protein that undergoes comprehensive conformational remodeling is [...] Read more.

In addition to the classic functions of proteins, such as acting as a biocatalyst or binding partner, the conformational states of proteins and their remodeling upon stimulation need to be considered. A prominent example of a protein that undergoes comprehensive conformational remodeling is transglutaminase 2 (TGase 2), the distinct conformational states of which are closely related to particular functions. Its involvement in various pathophysiological processes, including fibrosis and cancer, motivates the development of theranostic agents, particularly based on inhibitors that are directed toward the transamidase activity. In this context, the ability of such inhibitors to control the conformational dynamics of TGase 2 emerges as an important parameter, and methods to assess this property are in great demand. Herein, we describe the application of the switchSENSE^® principle to detect conformational changes caused by three irreversibly binding N^ε-acryloyllysine piperazides, which are suitable radiotracer candidates of TGase 2. The switchSENSE^® technique is based on DNA levers actuated by alternating electric fields. These levers are immobilized on gold electrodes with one end, and at the other end of the lever, the TGase 2 is covalently bound. A novel computational method is introduced for describing the resulting lever motion to quantify the extent of stimulated conformational TGase 2 changes. Moreover, as a complementary biophysical method, native polyacrylamide gel electrophoresis was performed under similar conditions to validate the results. Both methods prove the occurrence of an irreversible shift in the conformational equilibrium of TGase 2, caused by the binding of the three studied N^ε-acryloyllysine piperazides. Full article

(This article belongs to the Special Issue 21st Century Enzymology: Further Advances in Identification and Characterization of Enzymes)

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21st Century Enzymology: Further Advances in Identification and Characterization of Enzymes

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