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

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A special issue of International Journal of Molecular Sciences (ISSN 1422-0067). This special issue belongs to the section "Biochemistry".

Deadline for manuscript submissions: 10 November 2023 | Viewed by 6525

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Special Issue Editors

Dr. Erika A. Taylor

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Guest Editor

Department of Chemistry, Wesleyan University, Middletown, CT 06459, USA
Interests: enzyme functional assignment; enzymatic reaction mechanism determination; enzyme inhibitor discovery and design; protein dynamics; allosteric enzyme regulation

Dr. Andrew J. Andrews

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Guest Editor

Department of Cancer Biology, Fox Chase Cancer Center, Philadelphia, PA 19111, USA
Interests: enzyme mechanism; hysteresis; metabolism; assay development; LC-MS/MS; thermodynamics; chromatin; PTM incorporation and recognition; enzyme crosstalk; regulation

Special Issue Information

Dear Colleagues,

Enzymology is a central area of biochemistry, at the intersection of biology, chemistry, physics and more recently computational sciences. The study of enzymes builds from bioinformatic analyses to allow the definitive assignment of function of a gene and its corresponding protein. Enzyme kinetic and mechanistic analyses, aid in our understanding of reaction catalysis and metabolic pathways. These results when combined with enzyme structure and dynamical studies, provide the foundation for numerous applications from drug discovery and development efforts to bacterial biosynthesis to bioremediation.

This special edition will explore the identification of new enzymes and their characterization. Protein structural and dynamical studies to help understand the mode of action of enzymes are also invited, as are studies toward the development of enzyme inhibitors.

Dr. Erika A. Taylor
Dr. Andrew J. Andrews
Guest Editors

Manuscript Submission Information

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Submitted manuscripts should not have been published previously, nor be under consideration for publication elsewhere (except conference proceedings papers). All manuscripts are thoroughly refereed through a single-blind peer-review process. A guide for authors and other relevant information for submission of manuscripts is available on the Instructions for Authors page. International Journal of Molecular Sciences is an international peer-reviewed open access semimonthly journal published by MDPI.

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Keywords

enzyme functional assignment
drug discovery and development
enzyme kinetics
enzyme mechanisms
enzyme structure
enzyme dynamics
allosteric enzyme regulation
microbial biosynthesis and bioengineering

Published Papers (7 papers)

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Research

Open AccessArticle

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

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

Viewed by 488

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 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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Open AccessArticle

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

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

Cited by 1 | Viewed by 1110

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 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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Open AccessArticle

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

Tatyana I. Rakhmanova

Yevgeniya I. Shurubor

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Boris F. Krasnikov

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

Viewed by 744

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 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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Open AccessArticle

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

Claudia Sanfilippo

Federica Cernuto

and

Angela Patti

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

Viewed by 553

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 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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Open AccessArticle

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

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

Viewed by 785

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 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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Open AccessArticle

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

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

Viewed by 703

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 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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Open AccessArticle

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

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

Viewed by 1489

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 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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