International Journal of Molecular Sciences

Research

Jump to: Review

19 pages, 11189 KiB

Open AccessArticle

Mode of Metal Ligation Governs Inhibition of Carboxypeptidase A

by Jorge Antonio Amador Balderas, Frank Beierlein, Anselm H. C. Horn, Senta Volkenandt, Leon Völcker, Nikoo Mokhtari, Jules Cesar Epee Ndongue and Petra Imhof

Int. J. Mol. Sci. 2024, 25(24), 13725; https://doi.org/10.3390/ijms252413725 - 23 Dec 2024

Viewed by 792

Abstract

Carboxypeptidase is a Zn-dependent protease that specifically recognises and hydrolyses peptides with a hydrophobic side chain at the C-terminal residue. According to hydrolysis mechanisms proposed in the literature, catalysis requires a water molecule to be close to the Zn ion so as to [...] Read more.

Carboxypeptidase is a Zn-dependent protease that specifically recognises and hydrolyses peptides with a hydrophobic side chain at the C-terminal residue. According to hydrolysis mechanisms proposed in the literature, catalysis requires a water molecule to be close to the Zn ion so as to be activated as a nucleophile. Among small molecules that resemble the slowly hydrolysed Gly-Tyr peptide, which have been previously designed as inhibitors and characterised structurally, a variant with the terminal amino acid in a D-configuration has been the most effective. Our molecular dynamics simulations of carboxypeptidase complexed with different variants of those inhibitor ligands as well as variants of the Gly-Tyr peptide show that the strength of the inhibitory effect is not related to the binding strength of the ligand. Our data rather support an earlier notion that the inhibition is, at least partially, due to blocking a coordination site at the Zn ion by the ligand coordinating the metal ion in a bidentate fashion. Full article

(This article belongs to the Special Issue Mechanism of Enzyme Catalysis: When Structure Meets Function)

► Show Figures

Figure 1

16 pages, 3491 KiB

Open AccessArticle

Micellar Choline-Acetyltransferase Complexes Exhibit Ultra-Boosted Catalytic Rate for Acetylcholine Synthesis—Mechanistic Insights for Development of Acetylcholine-Enhancing Micellar Nanotherapeutics

by Davide Dante, Jatin Jangra, Anurag T. K. Baidya, Rajnish Kumar and Taher Darreh-Shori

Int. J. Mol. Sci. 2024, 25(24), 13602; https://doi.org/10.3390/ijms252413602 - 19 Dec 2024

Cited by 1 | Viewed by 708

Abstract

Choline-acetyltransferase (ChAT) is the key cholinergic enzyme responsible for the biosynthesis of acetylcholine (ACh), a crucial signaling molecule with both canonical neurotransmitter function and auto- and paracrine signaling activity in non-neuronal cells, such as lymphocytes and astroglia. Cholinergic dysfunction is linked to both [...] Read more.

Choline-acetyltransferase (ChAT) is the key cholinergic enzyme responsible for the biosynthesis of acetylcholine (ACh), a crucial signaling molecule with both canonical neurotransmitter function and auto- and paracrine signaling activity in non-neuronal cells, such as lymphocytes and astroglia. Cholinergic dysfunction is linked to both neurodegenerative and inflammatory diseases. In this study, we investigated a serendipitous observation, namely that the catalytic rate of human recombinant ChAT (rhChAT) protein greatly differed in buffered solution in the presence and absence of Triton X-100 (TX100). At a single concentration of 0.05% (v/v), TX100 boosted the specific activity of rhChAT by 4-fold. Dose–response analysis within a TX100 concentration range of 0.8% to 0.008% (accounting for 13.7 mM to 0.013 mM) resulted in an S-shaped response curve, indicative of an over 10-fold boost in the catalytic rate of rhChAT. This dramatic boost was unlikely due to a mere structural stabilization since it remained even after the addition of 1.0 mg/mL gelatin to the ChAT solution as a protein stabilizer. Furthermore, we found that the catalytic function of the ACh-degrading enzyme, AChE, was unaffected by TX100, underscoring the specificity of the effect for ChAT. Examination of the dose–response curve in relation to the critical micelle concentration (CMC) of TX100 revealed that a boost in ChAT activity occurred when the TX100 concentration passed its CMC, indicating that formation of micelle–ChAT complexes was crucial. We challenged this hypothesis by repeating the experiment on Tween 20 (TW20), another non-ionic surfactant with ~3-fold lower CMC compared to TX100 (0.06 vs. 0.2 mM). The analysis confirmed that micelle formation is crucial for ultra-boosting the activity of ChAT. In silico molecular dynamic simulation supported the notion of ChAT–micelle complex formation. We hypothesize that TX100 or TW20 micelles, by mimicking cell–membrane microenvironments, facilitate ChAT in accessing its full catalytic potential by fine-tuning its structural stabilization and/or enhancing its substrate accessibility. These insights are expected to facilitate research toward the development of new cholinergic-enhancing therapeutics through the formulation of micelle-embedded ChAT nanoparticles. Full article

(This article belongs to the Special Issue Mechanism of Enzyme Catalysis: When Structure Meets Function)

► Show Figures

Graphical abstract

18 pages, 2933 KiB

Open AccessArticle

Purification and Electron Transfer from Soluble c-Type Cytochrome TorC to TorA for Trimethylamine N-Oxide Reduction

by Alka Panwar, Berta M. Martins, Frederik Sommer, Michael Schroda, Holger Dobbek, Chantal Iobbi-Nivol, Cécile Jourlin-Castelli and Silke Leimkühler

Int. J. Mol. Sci. 2024, 25(24), 13331; https://doi.org/10.3390/ijms252413331 - 12 Dec 2024

Viewed by 729

Abstract

The enterobacterium Escherichia coli present in the human gut can reduce trimethylamine N-oxide (TMAO) to trimethylamine during anaerobic respiration. The TMAO reductase TorA is a monomeric, bis-molybdopterin guanine dinucleotide (bis-MGD) cofactor-containing enzyme that belongs to the dimethyl sulfoxide reductase family of molybdoenzymes. TorA [...] Read more.

The enterobacterium Escherichia coli present in the human gut can reduce trimethylamine N-oxide (TMAO) to trimethylamine during anaerobic respiration. The TMAO reductase TorA is a monomeric, bis-molybdopterin guanine dinucleotide (bis-MGD) cofactor-containing enzyme that belongs to the dimethyl sulfoxide reductase family of molybdoenzymes. TorA is anchored to the membrane via TorC, a pentahemic c-type cytochrome which receives the electrons from the menaquinol pool. Here, we designed an expression system for the production of a stable soluble form of multiheme-containing TorC, providing, for the first time, the purification of a soluble pentahemic cytochrome-c from E. coli. Our focus was to investigate the interaction between TorA and soluble TorC to establish the electron transfer pathway. We solved the X-ray structure of E. coli TorA and performed chemical crosslinking of TorA and TorC. Another goal was to establish an activity assay that used the physiological electron transfer pathway instead of the commonly used unphysiological electron donors methylviologen or benzylviologen. An AlphaFold model including the crosslinking sites provided insights into the electron transfer between TorC_C and the active site of TorA. Full article

(This article belongs to the Special Issue Mechanism of Enzyme Catalysis: When Structure Meets Function)

► Show Figures

Figure 1

26 pages, 7707 KiB

Open AccessArticle

Interaction of Tri-Cyclic Nucleobase Analogs with Enzymes of Purine Metabolism: Xanthine Oxidase and Purine Nucleoside Phosphorylase

by Alicja Stachelska-Wierzchowska, Marta Narczyk, Jacek Wierzchowski, Agnieszka Bzowska and Beata Wielgus-Kutrowska

Int. J. Mol. Sci. 2024, 25(19), 10426; https://doi.org/10.3390/ijms251910426 - 27 Sep 2024

Viewed by 1101

Abstract

Fluorescent markers play important roles in spectroscopic and microscopic research techniques and are broadly used in basic and applied sciences. We have obtained markers with fluorescent properties, two etheno derivatives of 2-aminopurine, as follows: 1,N²-etheno-2-aminopurine (1,N²-ε2APu, I) and [...] Read more.

Fluorescent markers play important roles in spectroscopic and microscopic research techniques and are broadly used in basic and applied sciences. We have obtained markers with fluorescent properties, two etheno derivatives of 2-aminopurine, as follows: 1,N²-etheno-2-aminopurine (1,N²-ε2APu, I) and N²,3-etheno-2-aminopurine (N²,3-ε2APu, II). In the present paper, we investigate their interaction with two key enzymes of purine metabolism, purine nucleoside phosphorylase (PNP), and xanthine oxidase (XO), using diffraction of X-rays on protein crystals, isothermal titration calorimetry, and fluorescence spectroscopy. Crystals were obtained and structures were solved for WT PNP and D204N-PNP mutant in a complex with N²,3-ε2APu (II). In the case of WT PNP—1,N²-ε2APu (I) complex, the electron density corresponding to the ligand could not be identified in the active site. Small electron density bobbles may indicate that the ligand binds to the active site of a small number of molecules. On the basis of spectroscopic studies in solution, we found that, in contrast to PNP, 1,N²-ε2APu (I) is the ligand with better affinity to XO. Enzymatic oxidation of (I) leads to a marked increase in fluorescence near 400 nm. Hence, we have developed a new method to determine XO activity in biological material, particularly suitable for milk analysis. Full article

(This article belongs to the Special Issue Mechanism of Enzyme Catalysis: When Structure Meets Function)

► Show Figures

Figure 1

20 pages, 5469 KiB

Open AccessArticle

Location Is Everything: Influence of His-Tag Fusion Site on Properties of Adenylosuccinate Synthetase from Helicobacter pylori

by Marija Zora Mišković, Marta Wojtyś, Maria Winiewska-Szajewska, Beata Wielgus-Kutrowska, Marija Matković, Darija Domazet Jurašin, Zoran Štefanić, Agnieszka Bzowska and Ivana Leščić Ašler

Int. J. Mol. Sci. 2024, 25(14), 7613; https://doi.org/10.3390/ijms25147613 - 11 Jul 2024

Cited by 2 | Viewed by 1328

Abstract

The requirement for fast and dependable protein purification methods is constant, either for functional studies of natural proteins or for the production of biotechnological protein products. The original procedure has to be formulated for each individual protein, and this demanding task was significantly [...] Read more.

The requirement for fast and dependable protein purification methods is constant, either for functional studies of natural proteins or for the production of biotechnological protein products. The original procedure has to be formulated for each individual protein, and this demanding task was significantly simplified by the introduction of affinity tags. Helicobacter pylori adenylosuccinate synthetase (AdSS) is present in solution in a dynamic equilibrium of monomers and biologically active homodimers. The addition of the His₆-tag on the C-terminus (C-His-AdSS) was proven to have a negligible effect on the characteristics of this enzyme. This paper shows that the same enzyme with the His₆-tag fused on its N-terminus (N-His-AdSS) has a high tendency to precipitate. Circular dichroism and X-ray diffraction studies do not detect any structural change that could explain this propensity. However, the dynamic light scattering, differential scanning fluorimetry, and analytical ultracentrifugation measurements indicate that the monomer of this construct is prone to aggregation, which shifts the equilibrium towards the insoluble precipitant. In agreement, enzyme kinetics measurements showed reduced enzyme activity, but preserved affinity for the substrates, in comparison with the wild-type and C-His-AdSS. The presented results reinforce the notion that testing the influence of the tag on protein properties should not be overlooked. Full article

(This article belongs to the Special Issue Mechanism of Enzyme Catalysis: When Structure Meets Function)

► Show Figures

Figure 1

14 pages, 4552 KiB

Open AccessArticle

Molecular Docking Studies of Ortho-Substituted Phenols to Tyrosinase Helps Discern If a Molecule Can Be an Enzyme Substrate

by María F. Montenegro, José A. Teruel, Pablo García-Molina, José Tudela, José Neptuno Rodríguez-López, Francisco García-Cánovas and Francisco García-Molina

Int. J. Mol. Sci. 2024, 25(13), 6891; https://doi.org/10.3390/ijms25136891 - 23 Jun 2024

Cited by 1 | Viewed by 1640

Abstract

Phenolic compounds with a position ortho to the free phenolic hydroxyl group occupied can be tyrosinase substrates. However, ortho-substituted compounds are usually described as inhibitors. The mechanism of action of tyrosinase on monophenols is complex, and if they are ortho-substituted, it is more [...] Read more.

Phenolic compounds with a position ortho to the free phenolic hydroxyl group occupied can be tyrosinase substrates. However, ortho-substituted compounds are usually described as inhibitors. The mechanism of action of tyrosinase on monophenols is complex, and if they are ortho-substituted, it is more complicated. It can be shown that many of these molecules can become substrates of the enzyme in the presence of catalytic o-diphenol, MBTH, or in the presence of hydrogen peroxide. Docking studies can help discern whether a molecule can behave as a substrate or inhibitor of the enzyme. Specifically, phenols such as thymol, carvacrol, guaiacol, eugenol, isoeugenol, and ferulic acid are substrates of tyrosinase, and docking simulations to the active center of the enzyme predict this since the distance of the peroxide oxygen from the oxy-tyrosinase form to the ortho position of the phenolic hydroxyl is adequate for the electrophilic attack reaction that gives rise to hydroxylation occurring. Full article

(This article belongs to the Special Issue Mechanism of Enzyme Catalysis: When Structure Meets Function)

► Show Figures

Figure 1

16 pages, 6234 KiB

Open AccessArticle

Structural Evaluation of a Nitroreductase Engineered for Improved Activation of the 5-Nitroimidazole PET Probe SN33623

by Abigail V. Sharrock, Jeff S. Mumm, Elsie M. Williams, Narimantas Čėnas, Jeff B. Smaill, Adam V. Patterson, David F. Ackerley, Gintautas Bagdžiūnas and Vickery L. Arcus

Int. J. Mol. Sci. 2024, 25(12), 6593; https://doi.org/10.3390/ijms25126593 - 15 Jun 2024

Viewed by 1474

Abstract

Bacterial nitroreductase enzymes capable of activating imaging probes and prodrugs are valuable tools for gene-directed enzyme prodrug therapies and targeted cell ablation models. We recently engineered a nitroreductase (E. coli NfsB F70A/F108Y) for the substantially enhanced reduction of the 5-nitroimidazole PET-capable probe, [...] Read more.

Bacterial nitroreductase enzymes capable of activating imaging probes and prodrugs are valuable tools for gene-directed enzyme prodrug therapies and targeted cell ablation models. We recently engineered a nitroreductase (E. coli NfsB F70A/F108Y) for the substantially enhanced reduction of the 5-nitroimidazole PET-capable probe, SN33623, which permits the theranostic imaging of vectors labeled with oxygen-insensitive bacterial nitroreductases. This mutant enzyme also shows improved activation of the DNA-alkylation prodrugs CB1954 and metronidazole. To elucidate the mechanism behind these enhancements, we resolved the crystal structure of the mutant enzyme to 1.98 Å and compared it to the wild-type enzyme. Structural analysis revealed an expanded substrate access channel and new hydrogen bonding interactions. Additionally, computational modeling of SN33623, CB1954, and metronidazole binding in the active sites of both the mutant and wild-type enzymes revealed key differences in substrate orientations and interactions, with improvements in activity being mirrored by reduced distances between the N5-H of isoalloxazine and the substrate nitro group oxygen in the mutant models. These findings deepen our understanding of nitroreductase substrate specificity and catalytic mechanisms and have potential implications for developing more effective theranostic imaging strategies in cancer treatment. Full article

(This article belongs to the Special Issue Mechanism of Enzyme Catalysis: When Structure Meets Function)

► Show Figures

Figure 1

12 pages, 2239 KiB

Open AccessArticle

Mechanistic and Structural Insights on Difluoromethyl-1,3,4-oxadiazole Inhibitors of HDAC6

by Edoardo Cellupica, Aureliano Gaiassi, Ilaria Rocchio, Grazia Rovelli, Roberta Pomarico, Giovanni Sandrone, Gianluca Caprini, Paola Cordella, Cyprian Cukier, Gianluca Fossati, Mattia Marchini, Aleksandra Bebel, Cristina Airoldi, Alessandro Palmioli, Andrea Stevenazzi, Christian Steinkühler and Barbara Vergani

Int. J. Mol. Sci. 2024, 25(11), 5885; https://doi.org/10.3390/ijms25115885 - 28 May 2024

Cited by 3 | Viewed by 1649

Abstract

Histone deacetylase 6 (HDAC6) is increasingly recognized for its potential in targeted disease therapy. This study delves into the mechanistic and structural nuances of HDAC6 inhibition by difluoromethyl-1,3,4-oxadiazole (DFMO) derivatives, a class of non-hydroxamic inhibitors with remarkable selectivity and potency. Employing a combination [...] Read more.

Histone deacetylase 6 (HDAC6) is increasingly recognized for its potential in targeted disease therapy. This study delves into the mechanistic and structural nuances of HDAC6 inhibition by difluoromethyl-1,3,4-oxadiazole (DFMO) derivatives, a class of non-hydroxamic inhibitors with remarkable selectivity and potency. Employing a combination of nuclear magnetic resonance (NMR) spectroscopy and liquid chromatography-mass spectrometry (LC-MS) kinetic experiments, comprehensive enzymatic characterizations, and X-ray crystallography, we dissect the intricate details of the DFMO-HDAC6 interaction dynamics. More specifically, we find that the chemical structure of a DMFO and the binding mode of its difluoroacetylhydrazide derivative are crucial in determining the predominant hydrolysis mechanism. Our findings provide additional insights into two different mechanisms of DFMO hydrolysis, thus contributing to a better understanding of the HDAC6 inhibition by oxadiazoles in disease modulation and therapeutic intervention. Full article

(This article belongs to the Special Issue Mechanism of Enzyme Catalysis: When Structure Meets Function)

► Show Figures

Figure 1

14 pages, 4056 KiB

Open AccessArticle

Nontraditional Roles of Magnesium Ions in Modulating Sav2152: Insight from a Haloacid Dehalogenase-like Superfamily Phosphatase from Staphylococcus aureus

by Jaeseok Bang, Jaehui Park, Sung-Hee Lee, Jinhwa Jang, Junwoo Hwang, Otabek Kamarov, Hae-Joon Park, Soo-Jae Lee, Min-Duk Seo, Hyung-Sik Won, Seung-Hyeon Seok and Ji-Hun Kim

Int. J. Mol. Sci. 2024, 25(9), 5021; https://doi.org/10.3390/ijms25095021 - 4 May 2024

Viewed by 1785

Abstract

Methicillin-resistant Staphylococcus aureus (MRSA) infection has rapidly spread through various routes. A genomic analysis of clinical MRSA samples revealed an unknown protein, Sav2152, predicted to be a haloacid dehalogenase (HAD)-like hydrolase, making it a potential candidate for a novel drug target. In this [...] Read more.

Methicillin-resistant Staphylococcus aureus (MRSA) infection has rapidly spread through various routes. A genomic analysis of clinical MRSA samples revealed an unknown protein, Sav2152, predicted to be a haloacid dehalogenase (HAD)-like hydrolase, making it a potential candidate for a novel drug target. In this study, we determined the crystal structure of Sav2152, which consists of a C2-type cap domain and a core domain. The core domain contains four motifs involved in phosphatase activity that depend on the presence of Mg²⁺ ions. Specifically, residues D10, D12, and D233, which closely correspond to key residues in structurally homolog proteins, are responsible for binding to the metal ion and are known to play critical roles in phosphatase activity. Our findings indicate that the Mg²⁺ ion known to stabilize local regions surrounding it, however, paradoxically, destabilizes the local region. Through mutant screening, we identified D10 and D12 as crucial residues for metal binding and maintaining structural stability via various uncharacterized intra-protein interactions, respectively. Substituting D10 with Ala effectively prevents the interaction with Mg²⁺ ions. The mutation of D12 disrupts important structural associations mediated by D12, leading to a decrease in the stability of Sav2152 and an enhancement in binding affinity to Mg²⁺ ions. Additionally, our study revealed that D237 can replace D12 and retain phosphatase activity. In summary, our work uncovers the novel role of metal ions in HAD-like phosphatase activity. Full article

(This article belongs to the Special Issue Mechanism of Enzyme Catalysis: When Structure Meets Function)

► Show Figures

Figure 1

Review

Jump to: Research

26 pages, 4932 KiB

Open AccessReview

Affinity Electrophoresis of Proteins for Determination of Ligand Affinity and Exploration of Binding Sites

by Patrick Masson and Tatiana Pashirova

Int. J. Mol. Sci. 2025, 26(7), 3409; https://doi.org/10.3390/ijms26073409 - 5 Apr 2025

Viewed by 279

Abstract

Affinity gel electrophoresis was introduced about 50 years ago. Proteins interact with a ligand immobilized in the support. Specific interactions cause a decrease in electrophoretic mobility. The presence of a free ligand, competing with an immobilized ligand, restores electrophoretic mobility. In affinity capillary [...] Read more.

Affinity gel electrophoresis was introduced about 50 years ago. Proteins interact with a ligand immobilized in the support. Specific interactions cause a decrease in electrophoretic mobility. The presence of a free ligand, competing with an immobilized ligand, restores electrophoretic mobility. In affinity capillary electrophoresis, the ligand is mobile, and its interaction with a specific protein changes the mobility of the protein–ligand complex. This review mostly focuses on gel affinity electrophoresis. The theoretical basis of this technique, ligand immobilization strategies, and principles for determination of ligand affinity are addressed. Factors affecting specificity and strength of interactions are discussed, in particular, the structure of the affinity matrix, pH, temperature, hydrostatic pressure, solvent, co-solvents, electric field, and other physico-chemical conditions. Capillary affinity electrophoresis principles and uses are also briefly introduced. Affinity gel electrophoresis can be used for qualitative and quantitative purposes. This includes detection of specific proteins in complex media, investigation of specific interactions, protein heterogeneity, molecular and genetic polymorphism, estimation of dissociation constants of protein–ligand complexes, and conformational stability of binding sites. Future prospects, in particular for screening of engineered mutants and potential new drugs, coupling to other analytical methods, and ultra-microtechnological developments, are addressed in light of trends and renewal of this old technique. Full article

(This article belongs to the Special Issue Mechanism of Enzyme Catalysis: When Structure Meets Function)

► Show Figures

Figure 1

26 pages, 8783 KiB

Open AccessReview

Intricate Structure–Function Relationships: The Case of the HtrA Family Proteins from Gram-Negative Bacteria

by Urszula Zarzecka and Joanna Skorko-Glonek

Int. J. Mol. Sci. 2024, 25(23), 13182; https://doi.org/10.3390/ijms252313182 - 7 Dec 2024

Viewed by 1361

Abstract

Proteolytic enzymes play key roles in living organisms. Because of their potentially destructive action of degrading other proteins, their activity must be very tightly controlled. The evolutionarily conserved proteins of the HtrA family are an excellent example illustrating strategies for regulating enzymatic activity, [...] Read more.

Proteolytic enzymes play key roles in living organisms. Because of their potentially destructive action of degrading other proteins, their activity must be very tightly controlled. The evolutionarily conserved proteins of the HtrA family are an excellent example illustrating strategies for regulating enzymatic activity, enabling protease activation in response to an appropriate signal, and protecting against uncontrolled proteolysis. Because HtrA homologs play key roles in the virulence of many Gram-negative bacterial pathogens, they are subject to intense investigation as potential therapeutic targets. Model HtrA proteins from bacterium Escherichia coli are allosteric proteins with reasonably well-studied properties. Binding of appropriate ligands induces very large structural changes in these enzymes, including changes in the organization of the oligomer, which leads to the acquisition of the active conformation. Properly coordinated events occurring during the process of HtrA activation ensure proper functioning of HtrA and, consequently, ensure fitness of bacteria. The aim of this review is to present the current state of knowledge on the structure and function of the exemplary HtrA family proteins from Gram-negative bacteria, including human pathogens. Special emphasis is paid to strategies for regulating the activity of these enzymes. Full article

(This article belongs to the Special Issue Mechanism of Enzyme Catalysis: When Structure Meets Function)

► Show Figures

Figure 1

33 pages, 2082 KiB

Open AccessReview

Applications of Microbial Organophosphate-Degrading Enzymes to Detoxification of Organophosphorous Compounds for Medical Countermeasures against Poisoning and Environmental Remediation

by Tatiana Pashirova, Rym Salah-Tazdaït, Djaber Tazdaït and Patrick Masson

Int. J. Mol. Sci. 2024, 25(14), 7822; https://doi.org/10.3390/ijms25147822 - 17 Jul 2024

Cited by 4 | Viewed by 2577

Abstract

Mining of organophosphorous (OPs)-degrading bacterial enzymes in collections of known bacterial strains and in natural biotopes are important research fields that lead to the isolation of novel OP-degrading enzymes. Then, implementation of strategies and methods of protein engineering and nanobiotechnology allow large-scale production [...] Read more.

Mining of organophosphorous (OPs)-degrading bacterial enzymes in collections of known bacterial strains and in natural biotopes are important research fields that lead to the isolation of novel OP-degrading enzymes. Then, implementation of strategies and methods of protein engineering and nanobiotechnology allow large-scale production of enzymes, displaying improved catalytic properties for medical uses and protection of the environment. For medical applications, the enzyme formulations must be stable in the bloodstream and upon storage and not susceptible to induce iatrogenic effects. This, in particular, includes the nanoencapsulation of bioscavengers of bacterial origin. In the application field of bioremediation, these enzymes play a crucial role in environmental cleanup by initiating the degradation of OPs, such as pesticides, in contaminated environments. In microbial cell configuration, these enzymes can break down chemical bonds of OPs and usually convert them into less toxic metabolites through a biotransformation process or contribute to their complete mineralization. In their purified state, they exhibit higher pollutant degradation efficiencies and the ability to operate under different environmental conditions. Thus, this review provides a clear overview of the current knowledge about applications of OP-reacting enzymes. It presents research works focusing on the use of these enzymes in various bioremediation strategies to mitigate environmental pollution and in medicine as alternative therapeutic means against OP poisoning. Full article

(This article belongs to the Special Issue Mechanism of Enzyme Catalysis: When Structure Meets Function)

► Show Figures

Figure 1

24 pages, 5865 KiB

Open AccessReview

4-Hydroxyphenylacetate 3-Hydroxylase (4HPA3H): A Vigorous Monooxygenase for Versatile O-Hydroxylation Applications in the Biosynthesis of Phenolic Derivatives

by Ping Sun, Shuping Xu, Yuan Tian, Pengcheng Chen, Dan Wu and Pu Zheng

Int. J. Mol. Sci. 2024, 25(2), 1222; https://doi.org/10.3390/ijms25021222 - 19 Jan 2024

Cited by 3 | Viewed by 2291

Abstract

4-Hydroxyphenylacetate 3-hydroxylase (4HPA3H) is a long-known class of two-component flavin-dependent monooxygenases from bacteria, including an oxygenase component (EC 1.14.14.9) and a reductase component (EC 1.5.1.36), with the latter being accountable for delivering the cofactor (reduced flavin) essential for o-hydroxylation. 4HPA3H has a [...] Read more.

4-Hydroxyphenylacetate 3-hydroxylase (4HPA3H) is a long-known class of two-component flavin-dependent monooxygenases from bacteria, including an oxygenase component (EC 1.14.14.9) and a reductase component (EC 1.5.1.36), with the latter being accountable for delivering the cofactor (reduced flavin) essential for o-hydroxylation. 4HPA3H has a broad substrate spectrum involved in key biological processes, including cellular catabolism, detoxification, and the biosynthesis of bioactive molecules. Additionally, it specifically hydroxylates the o-position of the C4 position of the benzene ring in phenolic compounds, generating high-value polyhydroxyphenols. As a non-P450 o-hydroxylase, 4HPA3H offers a viable alternative for the de novo synthesis of valuable natural products. The enzyme holds the potential to replace plant-derived P450s in the o-hydroxylation of plant polyphenols, addressing the current significant challenge in engineering specific microbial strains with P450s. This review summarizes the source distribution, structural properties, and mechanism of 4HPA3Hs and their application in the biosynthesis of natural products in recent years. The potential industrial applications and prospects of 4HPA3H biocatalysts are also presented. Full article

(This article belongs to the Special Issue Mechanism of Enzyme Catalysis: When Structure Meets Function)

► Show Figures

Figure 1

Journal Menu

Journal Browser

Mechanism of Enzyme Catalysis: When Structure Meets Function

Share This Special Issue

Special Issue Editor

Special Issue Information

Keywords

Benefits of Publishing in a Special Issue

Published Papers (13 papers)

Research

Review

Further Information

Guidelines

MDPI Initiatives

Follow MDPI